Recombinant Protein Purification Handbook PDF

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Recombinant Protein Purification Handbook – Principles and Methods

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18-1142-75 AC 01/2007
Handbooks
from GE Healthcare
Protein Purification GST Gene Fusion System

Handbook Handbook
18-1132-29 18-1157-58
Gel Filtration Hydrophobic Interaction and

Principles and Methods Reversed Phase Chromatography
18-1022-18 Principles and Methods
11-0012-69
Affinity Chromatography
Principles and Methods 2-D Electrophoresis
18-1022-29 using immobilized pH gradients
Antibody Purification 80-6429-60
Handbook
18-1037-46 Microcarrier Cell Culture
Percoll 18-1140-62
Methodology and Applications
18-1115-69 Challenging Protein Purification
Handbook
Ion Exchange Chromatography 28-9095-31
& Chromatofocusing
Principles and Methods Recombinant Protein
11-0004-21 Purification Handbook
18-1142-75
Recombinant Protein
Handbook 18-1142-75AC 1
Content
Introduction ...................................................................................................................................................... 5
Chapter 1
Expression and sample preparation...................................................................................................... 9
Components of the expression system ........................................................................................................................... 9
Sample preparation ................................................................................................................................................................ 12
Chapter 2
Manual and automated purification .................................................................................................. 19
Tagged recombinant proteins for simple purification ............................................................................................. 19
Manual purification techniques ......................................................................................................................................... 19
Automated purification using ÄKTAdesign chromatography systems ........................................................... 20
Chapter 3
Purification of histidine-tagged recombinant proteins .............................................................. 23
Introduction ................................................................................................................................................................................ 23
Expression ................................................................................................................................................................................... 23
Purification overview .............................................................................................................................................................. 23
Purification using Ni Sepharose High Performance ................................................................................................. 31
Purification using Ni Sepharose 6 Fast Flow ................................................................................................................ 34
High-throughput screening using His MultiTrap HP and His MultiTrap FF 96-well filter plates ........... 39
Minipreps using His SpinTrap ............................................................................................................................................. 43
Purification using HisTrap HP and HisTrap FF ............................................................................................................. 46
Purification using HisTrap FF with ÄKTAprime plus .................................................................................................. 52
Purification from unclarified cell lysate using HisTrap FF crude ........................................................................ 55
Purification using a syringe and HisTrap FF crude Kit ............................................................................................ 61
Gravity-flow purification using His GraviTrap and His GraviTrap Kit ............................................................... 66
Scale-up purification using HisPrep FF 16/10 ............................................................................................................. 70
Purification using uncharged media ............................................................................................................................... 72
Purification using IMAC Sepharose High Performance ........................................................................................... 74
Purification using IMAC Sepharose 6 Fast Flow ......................................................................................................... 77
Purification using HiTrap IMAC HP and HiTrap IMAC FF columns ...................................................................... 80
Preparative purification using HiPrep IMAC FF 16/10 column ............................................................................. 84
Detection of histidine-tagged proteins .......................................................................................................................... 88
Tag removal by enzymatic cleavage .............................................................................................................................. 91
Troubleshooting ........................................................................................................................................................................ 93
2 Handbook 18-1142-75AC
Chapter 4
Optimizing purification of histidine-tagged proteins .................................................................. 97
Introduction ................................................................................................................................................................................ 97
Optimizing using imidazole .................................................................................................................................................. 97
Optimizing using different metal ions .......................................................................................................................... 100
Optimizing using multistep purifications .................................................................................................................... 103
Chapter 5
Purification of GST-tagged recombinant proteins .................................................................... 105
Introduction ............................................................................................................................................................................. 105
Expression ................................................................................................................................................................................ 105
Purification ............................................................................................................................................................................... 108
Selecting a product for GST-tagged protein purification .................................................................................... 109
General considerations for purification of GST-tagged proteins .................................................................... 112
Selecting equipment for purification ............................................................................................................................ 113
Purification using Glutathione Sepharose High Performance,
Glutathione Sepharose 4 Fast Flow, and Glutathione Sepharose 4B ............................................................ 114
High-throughput screening using GST MultiTrap FF and GST MultiTrap 4B 96-well filter plates ...... 120
Minipreps using the GST SpinTrap Purification Module ....................................................................................... 125
Purification using GSTrap HP, GSTrap FF, and GSTrap 4B columns ................................................................ 126
Preparative purification using GSTPrep FF 16/10 column .................................................................................. 134
Troubleshooting of purification methods ................................................................................................................... 138
Detection of GST-tagged proteins ................................................................................................................................. 142
Troubleshooting of detection methods ....................................................................................................................... 151
Removal of GST tag by enzymatic cleavage ............................................................................................................ 153
Troubleshooting of cleavage methods ........................................................................................................................ 166
Chapter 6
Simple purification of other recombinant or native proteins ............................................... 169
Chapter 7
Multistep purification of tagged and untagged recombinant proteins ........................... 175
Chapter 8
Handling inclusion bodies .................................................................................................................... 185
Solubilization of inclusion bodies ................................................................................................................................... 185
Refolding of solubilized recombinant proteins ........................................................................................................ 186
Troubleshooting ..................................................................................................................................................................... 190
Chapter 9
Desalting and buffer exchange ......................................................................................................... 191
PD-10 Desalting ..................................................................................................................................................................... 193
HiTrap Desalting .................................................................................................................................................................... 194
HiPrep 26/10 Desalting ....................................................................................................................................................... 198
Appendix 1
Characteristics of Ni Sepharose and uncharged IMAC Sepharose products ................ 201
Ni Sepharose products ....................................................................................................................................................... 201
Uncharged IMAC Sepharose products ........................................................................................................................ 207
Appendix 2
Characteristics of Glutathione Sepharose products ................................................................ 211
Appendix 3
Precipitation and resolubilization...................................................................................................... 215
Appendix 4
Column packing and preparation ..................................................................................................... 219
Appendix 5
Conversion data: proteins, column pressures ............................................................................. 222
Appendix 6
Converting from linear flow (cm/h) to volumetric flow rates (ml/min)
and vice versa ............................................................................................................................................ 223
Appendix 7
GST vectors ................................................................................................................................................. 224
Control regions for pGEX vectors ................................................................................................................................... 225
Appendix 8
Amino acids table..................................................................................................................................... 226
Appendix 9
Principles and standard conditions for different purification techniques ...................... 228
Product index ............................................................................................................................................. 235
Related literature ...................................................................................................................................... 236
Ordering information .............................................................................................................................. 237
Introduction
This handbook is intended for those interested in the expression and purification of recombinant proteins.
The use of recombinant proteins has increased greatly in recent years, as has the wealth of techniques
and products used for their expression and purification. The advantages of using a protein/peptide tag
fused to the recombinant protein to facilitate its purification and detection is now widely recognized. In
some cases, tags may improve the stability and solubility of recombinant proteins.
The reader will be introduced to the initial considerations to be made when deciding upon host, vector,
and use of a tagged or untagged protein. General guidelines for successful protein expression are also
included. Advice is given on harvesting and extraction, handling of inclusion bodies, tag removal, and
removal of unwanted salts and small molecules.
Purification of recombinant proteins can be performed manually or by using a chromatography system.
The system can be operated manually or it can be automated to save time and effort. The purification
can be performed on many scales, in columns of various sizes. Columns can be purchased prepacked
with a chromatographic medium, or empty columns can be packed manually. Purification can also be
performed in batch, with gravity flow, in SpinTrap™ columns using centrifugation, or in a 96-well plate
format using MultiTrap™ products.
Proteins are purified using chromatography techniques that separate them according to differences in
their specific properties, as shown in Figure 1. Tags enable recombinant proteins to be purified by affinity
chromatography designed to capture the tagged recombinant protein based on biorecognition of the
tag. Thus, several different recombinant proteins can be purified by the same affinity technique if they
all have the same tag. In the same way, tags also allow the use of a common detection protocol for
different recombinant proteins. Consequently, tagged proteins are simple and convenient to work with
and, for many applications, a single purification step, using a commercially available chromatography
column, is sufficient. This is clearly demonstrated in the specific chapters on the expression, purification,
and detection of recombinant proteins fused with the commonly used histidine or glutathione S-
transferase (GST) tags. A scheme for the general purification of histidine-tagged proteins is given in
Figure 2. In addition, suggestions for the successful purification of untagged recombinant proteins by a
single affinity chromatography step are also given in this handbook. When a higher degree of purity is
required for either tagged or untagged recombinant proteins, a multistep purification will be necessary.
This can become a straightforward task by choosing the right combination of purification techniques.
Gel filtration Hydrophobic interaction Ion exchange Affinity Reversed phase
Fig 1. Separation principles in chromatographic purification.
In summary, this handbook aims to help the reader achieve a protein preparation that contains the
recombinant protein of interest in the desired quantity and quality required for their particular needs.
The quality of the recombinant protein can be reflected in its folding and biological activity.
Common acronyms and abbreviations
A280 UV absorbance at specified wavelength (in this example, 280 nanometers)
AC affinity chromatography
BCA bicinchoninic acid
CDNB 1-chloro-2,4-dinitrobenzene
CF chromatofocusing
CIPP Capture, Intermediate Purification, and Polishing
CV column volume
DAB 3,3'-diaminobenzidine
DNase deoxyribonuclease
ELISA enzyme-linked immunosorbent assay
FF Fast Flow
Gua-HCl guanidine-HCl
GF gel filtration
GST glutathione S-transferase
HIC hydrophobic interaction chromatography
HMW high molecular weight
HP High Performance
HRP horseradish peroxidase
IEX ion exchange chromatography
IMAC immobilized metal ion affinity chromatography
IPTG isopropyl β-D-thiogalactoside
LMW low molecular weight
MPa megaPascal
Mr relative molecular weight
N/m column efficiency expressed as theoretical plates per meter
PBS phosphate buffered saline
pI isoelectric point, the pH at which a protein has zero net surface charge
psi pounds per square inch
PMSF phenylmethylsulfonyl fluoride
PVDF polyvinylidene fluoride
r recombinant, as in rGST and rBCA
RNase ribonuclease
RPC reverse phase chromatography
SDS sodium dodecyl sulfate
SDS-PAGE sodium dodecyl sulfate polyacrylamide gel electrophoresis
TCEP Tris(2-carboxyethyl)phosphine hydrochloride
Symbols
this symbol indicates general advice to improve procedures or recommend action under
specific situations.
this symbol denotes mandatory advice and gives a warning when special care should be taken.
this symbol highlights troubleshooting advice to help analyze and resolve difficulties.
highlights chemicals, buffers and equipment.
outline of experimental protocol.
General purification of histidine-tagged proteins
Native Denaturing
conditions conditions
Binding buffer
Binding buffer (including 20 to
(including 20 to 40 mM imidazole
40 mM imidazole) Cell lysis and 8 M urea
or 6 M guanidine
hydrochloride)
Binding to
affinity media
Binding buffer
40 mM imidazole) Wash and 8 M urea
or 6 M guanidine
hydrochloride)
Elution buffer: Binding buffer Elution buffer: Binding buffer
with a higher concentration Elute with a higher concentration
of imidazole of imidazole
Pure tagged Pure denatured

protein tagged protein
Refolding
tagged protein
cell protein Pure tagged

protein
denatured tagged protein
Fig 2. General purification workflow for histidine-tagged proteins (assumes use of Ni2+-charged affinity media, but other metal-ion-
charged media follow the same workflow).
Chapter 1
Expression and sample preparation
Components of the expression system
A protein expression system includes, among other things, a vector with an appropriate promoter and
other regulatory sequences, along with the gene encoding the recombinant protein of interest. Vectors
are available commercially for the expression of recombinant proteins either fused to a tag or untagged.
Such expression vectors are designed with control regions to suit the specific host (for example, E. coli
versus mammalian cells) and type of expression needed. The presence of resistance markers makes
selection of the correct clones more straightforward. Expression of the recombinant protein can be
constitutive or regulated, or it can be at a high or low level, depending on the specific requirements.
The choice of vector is important because it affects so many of the processes that follow the cloning
steps including expression, protein processing, and purification. The completed vector construct is
used in a prokaryotic or eukaryotic organism, tissue, or cell line to produce the recombinant protein
that may be of academic and/or industrial importance. The recombinant protein may then need to be
detected, quantitated, and/or purified. Selection of a suitable expression system depends on the
desired scale of production, the time and resources available, and the intended use of the recombinant
protein. Several alternative systems for expression may be suitable.
Choice of host
Many host systems are available including bacteria, yeast, plants, filamentous fungi, insect or mammalian
cells grown in culture, and transgenic animals or plants. Each host system has its own advantages and
disadvantages, and it is important to consider these before final selection of host.
The choice of host affects not only the expression of the protein but also the way in which the product
can be subsequently purified. In order to decide which host is most suitable, the amount and the degree
of purity of the product, as well as its biological integrity and potential toxicity, should be considered.
For example, bacterial expression systems are not suitable if post-translational modification is
required to produce a fully functional recombinant product. Table 1 summarizes features of several
expression systems.
Table 1. Features of several types of expression systems.
Processing Bacteria Yeast Insect Mammalian

cells cells
Inclusion bodies +/- (+)/- – –
Secretion +/– +1 + +
Glycosylation – +2 + +
Proteolytic cleavage +/– +/– – –
Other post-translational – +3 + +
modifications
+ = Yes
– = No
1 Constructs are often prepared to allow secretion of the protein. This eliminates the need for cell lysis, which requires more powerful
methods for yeast than for E. coli.

2 Yeast give more extensive glycosylation than insect cells and mammalian cells; this is a drawback of heterologous expression in yeast.
3 Yeast lack some functions of post-translational modifications that exist in higher eukaryotes.
The location of product within the host will affect the choice of methods for isolation and purification of
the product. For example, in addition to expressing the protein cytoplasmically, a bacterial host may
secrete the protein into the growth medium, transport it to the periplasmic space, or store it as insoluble
inclusion bodies within the cytoplasm (Fig 3). Expression in different parts of the cell will lead to varying
amounts of cellular (contaminant) proteins that will need to be removed to obtain a pure target protein.
The main focus of this handbook is purification of soluble proteins from bacterial sources, as these are
the most common systems. Purification of proteins expressed as inclusion bodies is also discussed (see
Chapter 8).
Medium
~10 proteins
Lipopolysaccharide
70 Å
70 Å Outer membrane
Peptidoglycan
210 Å Periplasm
~100 proteins
70 Å Inner membrane
Cytoplasm
~2000 proteins
Fig 3. Schematic cross-section of the cell wall and typical number of protein species in E. coli.
Choice of vector
The choice of vector family is largely governed by the host. Once the host has been selected, many
different vectors are available for consideration, from simple expression vectors to those that contain
specialized sequences in order to secrete the recombinant proteins. In order to clone the gene of interest,
all engineered vectors have a selection of unique restriction sites downstream of a transcription
promoter sequence. Recent developments in cloning technology provide increased flexibility in the
choice of host and vector systems, including options allowing the DNA sequence of interest to be
inserted into multiple types of expression vectors.
The expression of a recombinant protein fused to a tag of known size and biological function can greatly
simplify subsequent purification and detection (for expression method development and purification).
In some cases, the protein yield can also be increased. Table 2 reviews some of the features of tagged
protein expression, purification, and detection that may influence the final choice of vector.
Table 2. Advantages and disadvantages of tagged versus untagged protein expression.
Advantages Disadvantages
Tagged proteins
Solubility and stability can be improved. Tag may interfere with protein structure and affect folding
Targeting information can be incorporated and biological activity.
into a tag. If tag needs to be removed, cleavage may not always be
A marker for expression is provided. achieved at 100%, and sometimes amino acids may be
left1.
Simple purification is possible using affinity
chromatography. Generic two-step purification
protocols can often be set up for lab-scale
protein production platforms.
Detection of the tag instead of the target protein
moiety allows for a generic detection method in,
e.g., protein production platforms for structural
biology.
Some tags allow strong binding to chromatography
media in the presence of denaturants, making
on-column refolding possible.
continues on following page
Table 2. Advantages and disadvantages of tagged versus untagged protein expression (continued).
Untagged proteins
Tag removal is not necessary. Purification and detection not as simple.
Problems with solubility and stability may be difficult to
overcome, reducing potential yield.
1The effectiveness of proteases used for cleavage may be decreased by substances, for example, detergents, in the protein preparation
or by inappropriate conditions.
Choice of tag
The two most commonly used tags are (histidine)6 and GST. Other polyhistidine tags consisting of
between 4 and 10 histidine residues are also used; they may provide useful alternatives to the
(histidine)6 tag if specific requirements exist for purification.
As for the selection of host and vectors, the decision to use either a histidine or a GST tag must be made
according to the requirements of the specific application. Table 3 highlights some key features of these
tags that should be considered.
Table 3. Choice of tag.
Histidine tag GST tag

Can be used in any expression system. Can be used in any expression system.
Purification procedure gives high yields of Purification procedure gives high yields of pure product.
pure product. The GST tag may also increase the solubility of expressed
proteins.
Selection of purification products available Selection of purification products available for any scale.
for any scale.
Small tag may not need to be removed (e.g., tag is Site-specific protease (PreScission™ Protease) enables
weakly immunogenic so fusion partner can be used highly specific cleavage at 4ºC. The protease is also easily
directly as an antigen in antibody production). removed because it is itself GST tagged (see Chapter 5).
Site-specific proteases enable cleavage of tag
if required.
TEV protease is often used to cleave off histidine
tags.
Note: Enterokinase sites that enable tag cleavage
without leaving behind extra amino acids are
preferable.
Histidine tag is easily detected using an GST tag easily detected using an enzyme or
immunoassay. immunoassay.
Simple purification. Simple purification. Very mild elution conditions minimize
Purification can be performed under denaturing risk of damage to functionality and antigenicity of target
conditions if required. Allows on-column refolding. protein. Buffer exchange may be desirable to remove
Note: Imidazole may cause precipitation in rare reduced glutathione.
cases. Buffer exchange to remove imidazole may
be necessary.
Histidine-dihydrofolate reductase tag stabilizes GST tag can help stabilize folding of recombinant proteins.
small peptides during expression.
Small tag is less likely to interfere with structure Tagged proteins form dimers.
and function of fusion partner.
GE Healthcare provides a variety of solutions for purification of histidine- and GST-tagged proteins.
Chapters 3 to 5 discuss these solutions in detail. GE Healthcare provides purification solutions for other
tagged proteins as well, including the calmodulin-binding peptide tag, the protein A tag, and biotinylated
peptide tags. Recombinant proteins fused to the calmodulin-binding peptide can be purified by
Calmodulin Sepharose™ 4B. Protein A-tagged proteins can be purified using IgG Sepharose Fast Flow.
Recombinant proteins with a biotinylated peptide tag can be purified using HiTrap™ Streptavidin HP
columns or by using Streptavidin Sepharose High Performance.
Sample preparation
The key to optimizing expression of tagged proteins is the capability to screen crude lysates from
many clones so that optimal expression levels and growth conditions can be readily determined. This
can easily be accomplished using the prepacked 96-well plates, His MultiTrap HP and His MultiTrap FF,
or GST MultiTrap 4B and GST MultiTrap FF (see Chapters 3 and 5). Once conditions are established, the
researcher is ready to prepare large-scale cultures of the desired clones. The samples are then processed
and prepared for purification. Various methods for the purification of tagged proteins are available,
depending on the expression system (host and vector) and the tag used. An overview of the sample
preparation process is depicted in Figure 4.
Intracellular Extracellular
expression expression
Insoluble in Soluble in Periplasmic Culture

cytoplasm cytoplasm space medium
Cell lysis Cell wall disruption
Cell debris removal Cell removal Cell removal
Harvest inclusion Recover Recover clarified Recover clarified

bodies supernatant sample sample
Solubilization
Purification
Fig 4. Overview of sample preparation.
Yield of recombinant proteins is highly variable and is affected by the nature of the tagged protein, the
host cell, and the culture conditions. Recombinant protein yields can range from 0 to 10 mg/l. Table 4
can be used to approximate culture volumes based on an average yield of 2.5 mg/l.
Table 4. Recombinant protein yields.
Protein 12.5 µg 50 µg 1 mg 10 mg 50 mg
Culture volume 5 ml 20 ml 400 ml 4l 20 l
Volume of lysate 0.5 ml 1 ml 20 ml 200 ml 1000 ml
For specific sample preparation steps, see Chapter 3 for histidine-tagged proteins and Chapter 5 for
GST-tagged proteins.
Cell harvesting and extraction
Cell harvesting and extraction procedures should be selected according to the source of the protein,
such as bacterial, plant, or mammalian, intracellular or extracellular. Harvesting, in which the cells are
separated from the cell culture media, generally involves either centrifugation or filtration. Refer to
standard protocols for the appropriate methodology based on the source of the target protein.
Selection of an extraction technique depends as much on the equipment available and scale of operation
as on the type of sample. Examples of common extraction processes for recombinant proteins are
shown in Table 5. In many situations, researchers may select a combination of these methods to
achieve optimal results.
Table 5. Common sample extraction processes for recombinant proteins.
Extraction process Typical conditions Comment

Gentle
Cell lysis (osmotic shock) 2 volumes water to 1 volume Lower product yield but reduced
packed prewashed cells. protease release.
Enzymatic digestion Lysozyme 0.2 mg/ml, Lab scale only, often combined
37°C, 15 min. with mechanical disruption.
Moderate
Grinding with abrasive, Add glass beads to prewashed Physical method. Chemical
e.g., glass beads cells, vortex, centrifuge, repeat up conditions are less important for
to five times, pooling supernatants. cell lysis but may be important
for subsequent removal of cell
debris and purification steps.
Freeze/thaw Freeze cells, thaw, resuspend pellet Several cycles.
by pipetting or gentle vortexing in
room-temperature lysis buffer.
Incubate, centrifuge, retain
supernatant.
Vigorous
Ultrasonication or bead milling Follow equipment instructions. Small scale; release of nucleic
acids may cause viscosity
problems (may add DNase to
decrease viscosity); inclusion
bodies must be resolubilized.
Manton-Gaulin homogenizer Follow equipment instructions. Large scale.
French press Follow equipment instructions. Lab scale.
Fractional precipitation See Appendix 3. Precipitates must be resolubilized.
The results obtained from cell lysis depend on several factors, including sample volume, cell concentration,
time, temperature, energy input (speed of agitation, pressure, etc.), and physical properties of the cell
lysis device.
Use procedures that are as gentle as possible because too vigorous cell or tissue disruption may
denature the target protein or lead to the release of proteolytic enzymes and general acidification.
Extraction should be performed quickly, at sub-ambient temperatures, in the presence of a

suitable buffer to maintain pH and ionic strength and stabilize the sample.
The release of nucleic acids may cause viscosity problems (addition of DNase may decrease
viscosity). Frequently, protease inhibitors are needed to reduce protein breakdown during
extraction. Fractional precipitation (see Appendix 3) may reduce the presence of proteases.
In bacterial and yeast expression systems, the recombinant protein may often be contained in
inclusion bodies. Extraction requires solubilization of the inclusion bodies, usually in the presence
of denaturants, followed by refolding before or after purification. Refer to Chapter 8 for more
information.
Preparation for chromatographic purification
Samples for chromatographic purification should be clear and free from particulate matter. Simple
steps to clarify a sample before beginning purification will avoid clogging the column, may reduce the
need for stringent washing procedures, and can extend the life of the chromatographic medium. An
exception to this rule is when purifying a histidine-tagged protein using HisTrap™ FF crude columns or
kit, His GraviTrap™ columns, His MultiTrap products (discussed in Chapter 3), or when purifying a GST-
tagged protein using GST MultiTrap products (discussed in Chapter 5). Use of any of these products
eliminates the need to clarify the sample and will therefore speed up the purification procedure. This
may be very important when purifying sensitive proteins, as a means to preserve their activity.
Major parameters to consider when preparing a sample for chromatographic purification include:
• Clarification (except for “crude” and GraviTrap columns as well as MultiTrap products; see above)
• Stabilization of target protein (protease inhibition, pH, ionic state, reducing agents, stabilizing
additives, etc.)
• Suitable conditions for chromatographic purification to work (mainly absorption, optimizing binding
of target protein and minimizing binding of contaminants)
• Available equipment
• Practicality and convenience (sample size, filtration/centrifugation equipment, etc.)
Protein stability
In the majority of cases, biological activity needs to be retained after purification. Retaining the activity
of the target molecule is also an advantage when following the progress of the purification, because
detection of the target molecule often relies on its biological activity. Denaturation of sample components
often leads to precipitation or enhanced nonspecific adsorption, both of which will impair column
function. Hence, there are many advantages to checking the stability limits of the sample and working
within these limits during purification.
Proteins generally contain a high degree of tertiary structure, kept together by van der Waals’ forces,
ionic and hydrophobic interactions, and hydrogen bonding. Any conditions capable of destabilizing
these forces may cause denaturation and/or precipitation. By contrast, peptides contain a low degree
of tertiary structure. Their native state is dominated by secondary structures, stabilized mainly by
hydrogen bonding. For this reason, peptides tolerate a much wider range of conditions than proteins.
This basic difference in native structures is also reflected in that proteins are not easily renatured,
while peptides often renature spontaneously. Protein quaternary structure and protein complexes
may pose additional challenges to a successful protein purification. Protein complexes are often held
together by weak interactions that require mild purification conditions, and perhaps removal of
incomplete species of the complex. Some proteins require coenzymes or cofactors to be active, and
membrane proteins may need lipids from their natural environment in the cell membrane to maintain
their native structure.
It is advisable to perform stability tests before beginning to develop a purification protocol. The list
below may be used as a basis for such testing:
• Test pH stability in steps of one pH unit between pH 2 and pH 9.
• Test salt stability with 0 to 2 M NaCl and 0 to 2 M (NH4)2SO4 in steps of 0.5 M (include buffering
agents as well).
• Test the temperature stability in 10°C steps from 4°C to 40°C. At a minimum, first test in the cold
room and at ambient temperature (22°C).
• Test the stability and occurrence of proteolytic activity by leaving an aliquot of the sample at room
temperature overnight. Centrifuge each sample, if possible, and measure activity and UV absorbance
at 280 nm in the supernatant. Run an SDS-polyacrylamide gel to check the size of the target protein.
Sometime taking a UV-VIS spectrum (190 to 800 nm) may be very useful (e.g., for cytochromes)
because active structure may be required for native spectra.
Sample clarification
Centrifugation and filtration are standard laboratory techniques for sample clarification and are used
routinely when handling small samples.
It is highly recommended to centrifuge and filter any sample immediately before chromatographic
purification, unless purifying a histidine-tagged protein using HisTrap FF crude columns or kit,
His GraviTrap columns, or His MultiTrap products (discussed in Chapter 3), or when purifying a
GST-tagged protein using GST MultiTrap products (discussed in Chapter 5).
A clarified sample that is not used immediately may within minutes start to precipitate. In this
situation, reclarification is recommended.
Keeping samples on ice until use is often recommended, even when purification is performed at
room temperature.
Centrifugation
Centrifugation removes most particulate matter, such as cell debris. If the sample is still not clear after
centrifugation, use filter paper or a 5 µm filter as a first step and one of the filters listed in Table 6 as a
second step.
For small sample volumes or proteins that adsorb to filters, centrifuge at 10 000 x g for 15 min.
For cell lysates, centrifuge at 40 000 to 50 000 x g for 30 min (may be reduced to 10 to 15 min if
speed is of the essence).
Serum samples can be filtered through glass wool after centrifugation to remove any remaining
lipids.
Use the cooling function of the centrifuge and precool the rotor by storing it in the cold room (or
by starting to cool the centrifuge well in advance with the rotor in place).
Filtration
Filtration removes particulate matter. Membrane filters that give the least amount of nonspecific binding
of proteins are composed of cellulose acetate or polyvinylidene fluoride (PVDF). For sample preparation
before chromatography, select a filter pore size in relation to the bead size of the chromatographic
medium as shown in Table 6.
Table 6. Selecting filter pore sizes.
Nominal pore size of filter Particle size of chromatographic medium

1 µm 90 µm and greater
0.45 µm 30 or 34 µm
0.22 µm 3, 10, 15 µm or when extra-clean samples or sterile filtration is required
Check the recovery of the target protein in a test run. Some proteins may adsorb nonspecifically
to filter surfaces.
Filters become “saturated” — that is, they have a certain capacity. It may be necessary to check
the capacity when setting up a protocol.
Desalting and buffer exchange
Desalting columns are suitable for many different sample volumes and will rapidly remove low-
molecular-weight contaminants in a single step at the same time as transferring the sample into the
correct buffer conditions. If desalting is the first chromatographic step, clarification will be needed.
Centrifugation and/or filtration of the sample before desalting is recommended. Detailed procedures
for buffer exchange and desalting are given in Chapter 9.
Dialysis and centrifugal ultrafiltration/concentration are also options for desalting and/or buffer
exchange, but the speed of using a desalting column makes it an especially attractive option.
The need for changed condition can sometimes be met simply by dilution (to reduce ion
strength), addition [to increase ammonium sulfate concentration for hydrophobic interaction
chromatography (HIC)], or titration to adjust pH.
At laboratory scale, when samples are reasonably clean after filtration or centrifugation, the buffer
exchange and desalting step can be omitted. For affinity chromatography or ion exchange chroma-
tography, it may be sufficient to adjust the pH of the sample and, if necessary, adjust the ionic
strength of the sample.
Rapidly process small or large sample volumes. Use before and/or between purification steps,
if needed (remember that each extra step can reduce yield and that desalting also dilutes the
sample).
Remove salts from proteins with molecular weight Mr > 5000.
Use 100 mM ammonium acetate or 100 mM ammonium hydrogen carbonate if volatile buffers
are required.
Detection and quantitation

Detection and quantitation of the target protein are needed when optimizing purification protocols. For
over-expressed proteins, the high concentration in itself can be used for detection of the target protein
fraction in a chromatogram, but in such a case verification of the identify of the protein in the final
preparation is needed. Specific detection of tagged proteins can often be accomplished by analyzing
the presence of the tag by activity or immunoassay, or simply by the spectral properties of the tag.
This may be especially important when multiple constructs with the same tag are prepared in high-
throughput platforms. Specific detection of the target protein can be obtained by functional assays,
immunodetection, and mass spectrometry. SDS-polyacrylamide gel electrophoresis (SDS-PAGE) is the
key method for checking purity of proteins. The target protein band can often be identified using the
apparent Mr obtained by including molecular weight markers in the analysis. Subsequent verification
of protein identity should always be obtained. Optimizing purification protocols may require functional
assays to assess the intactness of the target protein. Detection methods specific for histidine- and
GST-tagged proteins are discussed in Chapters 3 and 5, respectively. In general:
• The relative yield of tagged protein can often be determined by measuring the absorbance at 280 nm
(suitable for both histidine- and GST-tagged proteins), because the purity after a single purification
step is high, that is, most of the eluted material may be considered to be the target protein. The
extinction coefficient of the target protein will be needed. A good estimation may be obtained by
theoretical calculation from the amino acid composition of the protein.
• The yield of protein may also be determined by standard chromogenic methods (e.g., Lowry,
BCA™ protein assay, Bradford, etc.).
• Immunoassays can be used for quantitation if a suitable standard curve can be produced. In this
case, it is not necessary to purify the tagged protein so long as a purified standard is available.
Therefore, these techniques may be used for quantitation during protocol development. The
immunoassay technique is also particularly suitable for screening large numbers of samples when
a simple yes/no answer is required (e.g., when testing fractions from a chromatographic run).
Assessing protein expression

Suboptimal expression of the target protein can be addressed by various methods, based on the cause
of the problem. If no target protein is detected in the extract, this may mean that the insert has been
cloned in an incorrect reading frame. It is essential that the protein-coding DNA sequences are cloned
in the proper translational reading frame in the vector. The best way to verify that the insert is in-frame
is to sequence the cloning junctions.
If yield of the target protein is low, it may be because the culture conditions have not been optimized
for its expression. Investigate the effect of cell strain, medium composition, incubation temperature,
and induction conditions (if applicable). Exact conditions will vary for each tagged protein expressed.
With E. coli systems, analyze a small aliquot of an overnight culture by, for example, SDS-PAGE, and if
available, use an activity assay.
Generally, a highly expressed protein will be visible by Coomassie™ blue staining when 5 to 10 µl of an
induced culture whose A600 is ~1.0 is loaded on the gel. Nontransformed host E. coli cells and cells
transformed with the parental vector should be run in parallel as negative and positive controls,
respectively.
The presence of the tagged protein in a total cell extract and its absence from a clarified lysate may
indicate the presence of inclusion bodies. Check for inclusion bodies using light microscopy. They are
often visible as dense spots in the cells. Refer to Chapter 8 for information on handling inclusion bodies.
It is also worthwhile to check for expression by immunoblotting. Run an SDS-PAGE of induced cells and
transfer the proteins to a nitrocellulose or PVDF membrane (such as Hybond™-C or Hybond-P). Detect
tagged protein using anti-histidine or anti-GST antibody, as appropriate, or an antibody directed toward
the specific target protein. Some tagged proteins may be masked on SDS-PAGE by a bacterial protein
of approximately the same molecular weight. Immunoblotting can be used to identify tagged proteins
in these cases.
If the target protein is present in the post-lysate pellet, consider methods to enrich it. Alternatively,
choose to secrete the product or add a stabilizing tag.
If the target protein is adsorbed to cell debris, test extraction at varying ionic strengths and pH to
dissociate it.
Occasionally, a high basal level of expression is observed, and this may pose problems of its own (e.g.,
this is a major concern if the expressed protein is toxic). The cause may be a leaky promoter. Different
vector systems rely on different constitutive and induced promoters, thus the most straightforward
means of addressing this problem is to try another system. It is also possible that the vector is simply
not compatible with the expression host; trying another vector or host should alleviate this problem.
Various modifications to recombinant proteins can arise during growth, and these too may affect
expression levels. These modifications include aggregation; misfolding and random disulfide bridges;
deamidation of asparagine and glutamine; oxidation of methionine; proteolytic cleavage; and other
modifications such as glycosylation, phosphorylation, and acylation. Discussion of these modifications
is beyond the scope of this handbook, but a simple first approach to reducing or eliminating problems
relating to them is, as above, to investigate the effect of cell strain, medium composition, incubation
temperature, and induction conditions. Exact conditions will vary for each tagged protein expressed.
Analytical tools useful for determining if a recombinant protein is correctly expressed are summarized
in Table 7.
Table 7. Analytical tools for assessing protein expression.
Analytical tool Characteristic being assessed

SDS-PAGE and immunoblotting Size
Proteolytic cleavage
Native PAGE Aggregation
Isoelectric focusing (IEF) Heterogeneity
Tests for biological activity Stability at different pH, ionic strengths, protein concentrations,
detergent concentrations
N-terminal sequencing Heterogeneous N-terminus
Mass spectrometry Size, sequence hetereogeneities, post-translational heterogeneities,
chemical modifications of amino acid residues
C-terminal sequencing (difficult method Truncated forms
performed in specialized labs)
Chapter 2
Manual and automated purification
Recombinant proteins are needed for research and industrial purposes in different qualities (e.g., with
native structure or denatured) and quantities (from microgram to gram scales). One needs to choose a
purification method that will yield protein of a quality and quantity that fits the intended use. The number
of samples that must be purified is also an important consideration. It may be possible to save valuable
time and protein samples by investing in a chromatography system.
Tagged recombinant proteins for simple purification

When a recombinant protein is fused to a peptide or protein tag, such as histidine or glutathione
S-transferase (GST), the properties of the tag can be exploited for purification purposes. Affinity
chromatography methods have been developed for each of the commonly used tags, and there is
a good chance of a successful purification of a tagged protein in a single step.
Manual purification techniques

For small-scale purification of tagged proteins, a single affinity chromatography step with a simple
elution by a step gradient is usually sufficient. Manual purification can be performed in batch or by
using gravity-flow or spin columns, or 96-well plates.
When a tagged protein is purified by a batch method, the protein sample is added to a purification
medium usually in a disposable plastic tube. The medium is then washed and the tagged protein is
eluted. The batch method is suited to purification on a small scale.
A tagged protein can also be purified by simply passing the protein sample through a disposable
column prepacked with an appropriate medium. There are columns especially designed for use by
gravity flow, for example, for histidine-tagged proteins His GraviTrap. A 1-ml His GraviTrap column can
purify approximately 40 mg of a histidine-tagged protein. In addition, there are HiTrap columns suitable
for use with a syringe or peristaltic pump for both histidine- and GST-tagged proteins (HisTrap and
GSTrap™ columns, respectively). In general the binding capacity for a histidine-tagged protein using a
HisTrap column is at least 40 mg per ml of medium. HiTrap columns can also be connected to ÄKTAdesign™
chromatography systems (see the next section in this chapter). The different connections are easy to
make because HiTrap columns come with all necessary connectors included.
When purification can be performed on a small scale or for expression screening, 96-well plates or
prepacked spin columns are convenient. For both histidine- and GST-tagged proteins, prepacked 96-well
plates (MultiTrap) are available. Samples are pipetted into the prepacked wells of the plate, with wash
and elution by centrifugation or vacuum. Each well has a capacity to purify up to about 1 mg of histidine-
tagged protein (His MultiTrap) and 0.5 mg of GST-tagged proteins (GST MultiTrap). Using these plates,
96 samples can be processed simultaneously. When many plates require processing, a robotic system
can be used for plate handling.
Prepacked spin columns (SpinTrap) designed for use in a microcentrifuge can offer an alternative to
screening using 96-well plates. His SpinTrap is such a spin column designed for the rapid purification and
screening of histidine-tagged proteins. Each column has the capacity to purify approximately 750 µg
of histidine-tagged protein. The GST SpinTrap Purification Module includes prepacked spin columns for
purifying up to 400 µg of GST-tagged protein per column.
Automated purification using ÄKTAdesign
chromatography systems
A chromatography system should be used when reproducible results are important and when manual
purification becomes too time-consuming and inefficient. Manual purification can become inefficient
when processes have to be repeated to obtain enough purified sample, when large sample volumes
have to be handled or when there are many different samples to be purified. In addition, the quality and
reproducibility of protein separations can be improved by using a chromatography system. Systems
provide more control than manual purification because of the ability to automatically monitor the
progress of the purification. Systems are robust and convenient to use. Not only can systems perform
simple step-gradient elution, but they can also provide high-resolution separations using accurately
controlled linear-gradient elution. They can work at the high flow rates of modern media. Following is a
description of the use of ÄKTAdesign chromatography systems suited to purification of tagged proteins.
ÄKTAprime™ plus (Fig 5) is an economical and easy-to-learn system for the purification of tagged
proteins. With push button control, it offers simple one-step purification of proteins. This system includes
preprogrammed methods for the purification of histidine- and GST- tagged proteins. In fact, there are
preprogrammed methods for the use of any HiTrap column. In addition, recovery of the recombinant
protein is often better than when the same protein is purified manually. With optimized purification
protocols and prepacked columns, yields and purity are highly consistent. Together with the appropriate
columns, tagged proteins can be purified in a single chromatography step on ÄKTAprime plus from
microgram to gram scale.
Fig 5. ÄKTAprime plus. Fig 6. ÄKTAexplorer.
Purification of tagged proteins can also be performed on more advanced chromatography systems.
ÄKTAexplorer™ (Fig 6) is a system where multiple samples (up to eight) of a tagged protein can be
automatically purified in a single step. This is very convenient because manual work between samples
is eliminated. Like ÄKTAprime plus, ÄKTAexplorer is a chromatography system that allows easy
purification of proteins from microgram to gram scale.
Another advantage offered by ÄKTAexplorer is that multiple samples of tagged proteins can be purified
automatically in multiple chromatography steps with the add-on ÄKTA™ 3D plus Kit. Using more than a
single chromatography step is important when a single affinity step does not yield the purity required
for a specific application or when a buffer-exchange or polishing step is needed after the affinity step.
When using the kit together with ÄKTAexplorer 100, up to six samples of tagged proteins can be
automatically purified in a single run, with protocols containing one or two steps. When a protocol with
three steps is selected, up to four samples can be purified. Often affinity chromatography (AC) is the
first step, and some protocols have a second purification step, gel filtration (GF), or ion exchange (IEX).
For added convenience and reproducibility, the purification protocols use recommended prepacked
columns. This system is capable of producing up to 50 mg of tagged protein of greater than 90% purity
per sample. These proteins are useful in structural and functional studies or in drug target screening.
ÄKTAxpress™ (Fig 7) is recommended when higher automation is required. ÄKTAxpress is a modular
system (from 1 to 12 modules controlled by one computer) for automated parallel purification of up to
48 samples of tagged proteins with purification protocols containing up to four steps. The purification
protocols may begin with AC followed by other purification steps such as desalting, IEX, and GF. In
addition, automatic on-column or off-column tag-removal steps can be integrated in the purification
protocol. All modules can work on the same protocol, or each module can work independently. The
purification protocols use prepacked columns and deliver purified samples of up to 50 mg of tagged
protein of > 95% purity. These purified samples are suitable, for example, for use in structural studies.
Fig 7. Four modules of ÄKTAxpress system.
There are other ÄKTAdesign systems available that can also be used for the purification of tagged
proteins. Standard ÄKTAdesign configurations are given in Fig 8.
More details about methods for purification are given in Chapter 3 and 4 for histidine-tagged proteins
and Chapter 5 for GST-tagged proteins.
Table 8. Ways of working with standard ÄKTAdesign configurations.
Standard ÄKTAdesign configurations

ÄKTA ÄKTA ÄKTA ÄKTA ÄKTA ÄKTA ÄKTA
Way of working prime plus purifier™ explorer xpress pilot™ crossflow™ process™
Manufacturing and production • •
UNICORN™ software • • • • • •
One-step simple purification • •
Reproducible performance • • • • • • •
for routine purification
System control and data • • • • • •
handling for regulatory
requirements
Automatic method development • • • •
and optimization
Automatic buffer preparation • •
Automatic pH scouting • •
Automatic media • •
or column scouting
Automatic multistep purification • •
Method development • • •
and scale-up
Sanitary design cGMP • • •
Scale-up, process • •
development, and
transfer to production
ÄKTAprime plus ÄKTApurifier ÄKTAexplorer ÄKTAxpress
ÄKTApilot ÄKTAcrossflow ÄKTAprocess

Fig 8. The standard ÄKTAdesign configurations.
Chapter 3
Purification of histidine-tagged recombinant
proteins
Introduction
Histidine-tagged proteins have a high selective affinity for Ni2+ and several other metal ions that can
be immobilized on chromatographic media using chelating ligands. Consequently, a protein containing
a histidine tag will be selectively bound to metal-ion-charged media such as Ni Sepharose High
Performance (HP) and Ni Sepharose 6 Fast Flow (FF) while other cellular proteins will not bind or bind
weakly. This chromatographic technique is often termed immobilized metal ion affinity chromatography
(IMAC). In general, the histidine-tagged protein is the strongest binder among all the proteins in a crude
sample extract (from, for example, a bacterial lysate). Eukaryotic extracts often have slightly more proteins
that can bind. Moreover, histidine tags are small and generally less disruptive than other tags to the
properties of the proteins on which they are attached. Because of this, tag removal may not always be
a priority.
Histidine-tagged protein expressed in E. coli can accumulate in two main forms, as biologically functional
soluble proteins or as inclusion bodies. Inclusion bodies are insoluble aggregates of denatured or partly
denatured protein that lack biological activity but often give a high yield of the recombinant protein.
To restore biological function of proteins expressed as inclusion bodies, solubilization, refolding, and
purification are necessary. This topic is discussed in more detail in Chapter 8.
Expression
General considerations for the expression of tagged proteins are discussed in Chapter 1, as are the
factors that should be considered when selecting the vector and host.
Purification overview
Figure 9 gives an overview of a typical purification workflow for histidine-tagged proteins, including
purification under denaturing conditions. On-column refolding and purification of histidine-tagged
proteins are also discussed in Chapter 8.
For simple, one-step purification of histidine-tagged proteins, a range of products is available designed
to meet specific purification needs. These products can be used for the purification of proteins containing
polyhistidine tags of different lengths (four to 10 histidine residues). A tag that is six residues long
(histidine)6 is most common. Under the standard binding and elution conditions described in this handbook,
a shorter tag, for example, (histidine)4, will bind more weakly and a longer (histidine)10 will bind more
strongly as compared with (histidine)6. This difference in binding strength can be used to advantage
during purification. For example, because a longer tag binds more strongly, a higher concentration of
imidazole can be included in the sample during loading (to prevent unwanted host cell proteins from
binding) as well as be used during the washing step before elution. This can facilitate the removal of
contaminants that may otherwise be copurified with a shorter tagged protein. For information on
optimizing protein purification of histidine-tagged proteins, refer to Chapter 4.
General purification of histidine-tagged proteins
Native Denaturing
conditions conditions
Binding buffer
40 mM imidazole) Cell lysis and 8 M urea
or 6 M guanidine
hydrochloride)
Binding to
affinity media
Binding buffer
40 mM imidazole) Wash and 8 M urea
or 6 M guanidine
hydrochloride)
Elution buffer: Binding buffer Elution buffer: Binding buffer
with a higher concentration Elute with a higher concentration
of imidazole of imidazole
Pure tagged Pure denatured

protein tagged protein
Refolding
tagged protein
cell protein Pure tagged

protein
denatured tagged protein
Fig 9. General purification workflow for histidine-tagged proteins (assumes use of Ni2+-charged affinity media, but other metal-ion-
charged media follow the same workflow).
General considerations
Types of media and formats
Media for purifying histidine-tagged proteins are available precharged with Ni2+ ions as well as uncharged.
Uncharged media can be charged with different metal ions in order to adjust selectivity. Charged
media from GE Healthcare include Ni Sepharose High Performance and Ni Sepharose 6 Fast Flow in
lab packs and prepacked formats. Uncharged media include IMAC Sepharose High Performance and
IMAC Sepharose 6 Fast Flow in lab packs and prepacked formats.
Ni Sepharose High Performance and Ni Sepharose 6 Fast Flow consist of highly cross-linked agarose
beads with an immobilized chelating group. As the product names indicate, the media are precharged
with Ni2+ ions. The media are compatible with all commonly used aqueous buffers, reducing agents,
denaturants such as 6 M guanidine-HCl (Gua-HCl) and 8 M urea, and a range of additives commonly
used in protein purification. Refer to Appendix 1 for a list of characteristics of the media.
Different sizes and types of prepacked columns and 96-well filter plates together with easily packed
Ni Sepharose High Performance and Ni Sepharose 6 Fast Flow bulk media (lab packs) provide fast,
convenient alternatives to the traditional batch method of protein purification. Batch preparations are
occasionally used if it appears that the tag is not fully accessible or when the protein in the lysate is at
very low concentrations (both could appear to give a low yield from the first purification step). A more
convenient alternative to improve yield is to decrease the flow rate or pass the sample through the
column several times.
We recommend always trying the precharged Ni Sepharose High Performance or Ni Sepharose 6 Fast
Flow media first. If you determine that increased selectivity would be advantageous, next try applying
other metal ions to one of the uncharged media. Test more than one metal ion to determine the one
best suited for your separation. GE Healthcare offers several uncharged IMAC purification products for
such purposes: convenient, prepacked 1-ml and 5-ml HiTrap IMAC HP and HiTrap IMAC FF and 20-ml
HiPrep™ IMAC FF 16/10 columns, as well as IMAC Sepharose High Performance and IMAC Sepharose 6
Fast Flow bulk media. Refer to Appendix 1 for a list of characteristics of the media.
Monitor purification steps by one or more of the detection methods referred to later in this chapter.
The choice of purification equipment should also be made according to the needs of the purification
(see Chapter 2).
Metal ion
In general, Ni2+ is the preferred metal for purification of recombinant histidine-tagged proteins. Note,
however, that in some cases it may be wise to test other metal ions, for example Zn2+ and Co2+, as the
strength of binding depends on the nature of the histidine-tagged protein as well as the metal ion. This
topic is also discussed in Chapter 4.
Leakage of Ni2+ from Ni Sepharose Fast Flow and High Performance is low under all normal
conditions. The leakage is lower than for other precharged IMAC media tested (see GE Healthcare
Data File 11-0008-86). In addition, leakage of metal ions from IMAC Sepharose High Performance
and IMAC Sepharose 6 Fast Flow is lower under normal conditions than is the case with other
IMAC media tested. For very critical applications, leakage during purification can be reduced
even further by performing a blank run before loading the sample (see purification procedures).
Working with nickel-containing products may produce an allergic reaction.
Buffers
Water and chemicals used for buffer preparation should be of high purity. Filter buffers through
a 0.22 µm or 0.45 µm filter before use.
We recommend use of the His Buffer Kit (available separately) to eliminate time-consuming
buffer preparation, thus promoting fast, reproducible, and convenient purification work. The kit
contains phosphate buffer concentrates and highly pure 2 M imidazole stock solution optimized
for rapid purification of histidine-tagged proteins.
We recommend binding at neutral to slightly alkaline pH (pH 7 to 8) in the presence of 0.5 to

1.0 M NaCl. Including salt in the buffers and samples eliminates ion-exchange effects but can
also have a marginal effect on the retention of proteins.
Sodium phosphate buffers are often used. Tris-HCl can generally be used, but should be avoided
in cases where the metal-protein affinity is weak, because it may reduce binding strength. Avoid
chelating agents such as EDTA or citrate in buffers. Imidazole is usually used for elution of histidine-
tagged proteins due to its efficiency at replacing the histidine tag by also interacting with the
metal ion. Low concentrations of imidazole should be used to wash out more weakly bound
host cell proteins to increase the purity of the target protein. Use highly pure imidazole, which
gives essentially no absorbance at 280 nm.
Membrane proteins must be purified in the present of a detergent in the sample and buffers. Notice
that the NaCl concentration may have to be optimized to avoid precipitation. Proteins expressed as
inclusion bodies can be solubilized in denaturants such as 8 M urea or 6 M Gua-HCl. The solubilized and
denatured protein can then be purified in the presence of the denaturant. If on-column refolding is to
be performed, an eluent with low concentration (or zero concentration) should be prepared. Refer to
Chapter 8 for a discussion of working with inclusion bodies.
Samples containing urea can be analyzed directly by SDS-PAGE whereas samples containing
Gua-HCl must be buffer-exchanged to a buffer with urea before SDS-PAGE, due to the high ionic
strength of Gua-HCl solutions.
Imidazole
Imidazole competes with proteins for binding to Ni Sepharose and IMAC Sepharose. Equilibration buffer
(binding and wash buffer) and sample should be complemented with a low concentration of imidazole to
reduce nonspecific binding of host cell proteins. The initial low concentration of imidazole establishes a
counter-ligand to the immobilized metal ion, which is important for controlled chromatography. At
somewhat higher concentrations, imidazole may also decrease the binding of histidine-tagged proteins.
The imidazole concentration in each step must therefore be optimized to ensure the best balance of
high purity (low binding of host cell proteins) and high yield (strong binding of histidine-tagged target
protein). The concentration of imidazole in the binding buffer and sample that will give optimal purifi-
cation results is protein dependent, and is usually slightly higher (20 to 40 mM is recommended) for
Ni Sepharose 6 Fast Flow and Ni Sepharose High Performance than for similar media on the market
(see GE Healthcare Data File 11-0008-86 for a discussion of this topic). See Chapter 4 for a discussion
on optimizing purification of histidine-tagged proteins by altering the imidazole concentration.
Use high-purity imidazole as this will give very low or no absorbance at 280 nm.
If imidazole needs to be removed from the protein, use a HiTrap Desalting, PD-10 Desalting, or
HiPrep 26/10 Desalting column depending on the sample volume.
Alternative elution solutions

As alternatives to imidazole elution, histidine-tagged proteins can be eluted by other methods or
combinations of methods; for example, lowering of pH within the range of 2.5 to 7.5 can be used.
Below pH 4, metal ions will be stripped off the medium.
Note: It is not always possible to elute with lower pH when using a metal ion other than Ni2+. This is
protein and metal ion dependent.
EGTA and EDTA, which are strong chelating compounds, can also be used for elution, but they will strip
the metal ions from the medium and thereby cause protein elution. The co-eluted metal ions will remain
chelated in the protein solution, but are easily removed with a desalting column, such as HiTrap
Desalting, PD-10 Desalting, or HiPrep 26/10 Desalting.
Note: The column needs to be recharged with metal ions before the next purification.
General procedure for sample preparation

For optimal conditions for growth, induction, and cell lysis of recombinant histidine-tagged proteins,
please refer to established procedures. The following is a general procedure for sample preparation
and cell lysis from bacterial cultures. Other established procedures may also work.
This procedure works well with the majority of the purification protocols included in this chapter.
However, some modifications of the procedures are noted where relevant.
1 Harvest cells from the culture by centrifugation at 7000 to 8000 × g for 10 min or at 1000 to 1500 × g for
30 min at 4°C.
2. Discard the supernatant. Place the bacterial pellet on ice.
3. Dilute cell paste (bacterial pellet) by adding 5 to 10 ml of binding buffer for each gram of cell paste.
To prevent the binding of host cell proteins with exposed histidines, it is essential to include
imidazole at a low concentration in the sample and binding buffer (see Chapter 4).
4a. Enzymatic lysis: Add 0.2 mg/ml lysozyme, 20 µg/ml DNase, 1 mM MgCl2, 1 mM Pefabloc™ SC or
phenylmethylsulfonyl fluoride (PMSF) (final concentrations). Stir for 30 min at room temperature or 4°C,
depending on the sensitivity of the target protein.
4b. Mechanical lysis: Disrupt cells by sonication on ice for approximately 10 min (in several short bursts), by
homogenization with a French press (or other homogenizer), or by freezing/thawing at least five times.
Mechanical lysis time may have to be extended to obtain an optimized lysate for sample loading
to avoid problems with back pressure. This is important when direct loading of unclarified, crude
sample without any clarification is performed (using HisTrap FF crude columns). Different proteins
have different sensitivity to cell lysis, and caution should be exercised to avoid heating and
frothing of the sample. If the sonicated or homogenized unclarified cell lysate is frozen before
use, precipitation and aggregation may increase. Additional sonication of the lysate can then
prevent increased back-pressure problems when loading on the column.
5. Measure and adjust pH if needed.
Do not use strong bases or acids for pH adjustment, as this may increase the risk of precipitation.
The sample should be fully dissolved. To avoid column clogging, we recommend centrifugation and
filtration through a 0.45 µm or 0.22 µm filter to remove cell debris or other particulate material.
Note: this is NOT necessary when using HisTrap FF crude, His GraviTrap, His MultiTrap HP,
or His MultiTrap FF.
If the sample is prepared in a buffer other than 20 mM phosphate buffer, 0.5 M NaCl, pH 7.4,
adjust its NaCl concentration to 0.5 M and pH to 7 to 8. This can be achieved by addition of
concentrated stock solutions, by dilution with the binding buffer, or by buffer exchange (on HiTrap
Desalting, PD-10 Desalting, or HiPrep 26/10 Desalting column, depending on the sample volume).
IMPORTANT! To minimize binding of host cell proteins, the sample should have the same
concentration of imidazole as the binding buffer. The concentration of imidazole is protein
dependent and should be determined empirically. We recommend starting with 20 to
40 mM imidazole.
If the recombinant histidine-tagged protein is expressed as inclusion bodies, they must be

solubilized using 6 M Gua-HCl or 8 M urea, and the chosen denaturant must be present in all
buffers during chromatography. Advice for working with inclusion bodies can be found in
Chapter 8 and in the troubleshooting section later in this chapter.
Purification using precharged media
Selection Guide — Precharged Ni Sepharose products
Will you use

Precharged an automated What
Ni Sepharose purification system Yes are your
such as requirements
media ÄKTAdesign ?
?
Batch Ni Sepharose 6 Fast Flow
Syringe 96-well His MultiTrap FF

No
plate His MultiTrap HP
Spin His SpinTrap

column
Buffers
included
? Yes His GraviTrap Kit
No
Gravity- Buffers
Yes flow included
HisTrap FF crude column
?
HisTrap FF No His GraviTrap
HisTrap FF crude Kit Contains Ni Sepharose High Performance.

Contains Ni Sepharose 6 Fast Flow.
Fig 10. Selection guide for the precharged Ni Sepharose products.
Figure 10 provides a selection guide for the precharged Ni Sepharose products, and Table 9 describes
these options in more detail. In general, Ni Sepharose High Performance is recommended when high
resolution and high capacity are important, whereas Ni Sepharose 6 Fast Flow is recommended when
scale-up is required. Similar information for the uncharged media follows later in this chapter, starting
on page 72.
Table 9. Purification options for histidine-tagged proteins using precharged media.
Product Format Approx. Description High- Mini- Batch/ Syr- ÄKTAdesign

or protein through- preps gravity inge system
column binding put flow
size capacity screening
Ni 25 ml 40 mg/ml For high resolution and + (+) - - +
Sepharose 100 ml elution of a more
High Per- concentrated sample
formance (high-performance
purification).
Yes HisTrap HP
High Prepacked
resolution columns
?
Ni Sepharose
No
High Performance
Scalability/ Prepacked
high flow columns No Ni Sepharose 6 Fast Flow
rates
?
Yes
Amount
of histidine-
>200 mg HisPrep FF 16/10
tagged
protein
Unclarified HisTrap FF crude
Clarified
<200 mg or unclarified
sample
Clarified HisTrap FF
Table 9. Purification options for histidine-tagged proteins using precharged media (continued).

HisTrap HP 1 ml 40 mg/ For use mainly with a - - - (+) +
column peristaltic pump or
5 ml 200 mg/ chromatography system.
column For high resolution and
elution of a more
concentrated sample
(high-performance
purification).
His 100 µl 0.75 mg/ For simple minipreps of - + - - -
SpinTrap column histidine-tagged proteins
and rapid expression
screening.
His 96-well 1 mg/ For high-throughput + - - - -
MultiTrap filter well screening. Can use with
HP plates robotics or manually
by centrifugation or
vacuum.
Table 9. Purification options for histidine-tagged proteins using precharged media (continued).

Ni 5 ml 40 mg/ml Excellent for scale-up + + + - +
Sepharose 25 ml due to high capacity
6 Fast Flow 100 ml and high flow properties.
500 ml
HisPrep™ FF 20 ml 800 mg/ For use with a - - - - +
16/10 column chromatography system.
Scale-up purification.
HisTrap FF 1 ml 40 mg/ For use with syringe, - - - + +
column peristaltic pump, or
5 ml 200 mg/ chromatography system.
column Provides excellent flow
properties.
Scale-up purification.
HisTrap FF 1 ml 40 mg/ For use with uncentrifuged, - - - + +
crude column crude cell lysates.
5 ml 200 mg/ For use with syringe,
column peristaltic pump, or
chromatography system.
HisTrap FF 3 × 1 ml 40 mg/ Kit includes 3 × 1 ml - - - + -
crude Kit column HisTrap FF crude columns,
all necessary buffers,
connectors, a syringe,
and instructions.
His 1 ml 40 mg/ For use with gravity flow, - - + - -
GraviTrap column allows direct purification
and His of both clarified and
GraviTrap unclarified cell lysates.
Kit Kit includes columns and
His Buffer Kit.
His 96-well 0.8 mg/ For high-throughput + - - - -
MultiTrap filter well screening. Can use with
FF plates robotics or manually
by centrifugation or
vacuum.
Companion product
His Buffer 1 kit N/A Premade buffers for manual - + + + -
Kit purification of histidine-
tagged proteins.
Contains Ni Sepharose 6 Fast Flow
Contains Ni Sepharose High Performance
Purification using Ni Sepharose High Performance
Ni Sepharose High Performance consists of highly cross-linked 6% agarose beads (34-µm diameter) to
which a chelating group has been immobilized and subsequently charged with Ni2+ ions. The chelating
group charged with Ni2+ provides very high binding capacity for histidine-tagged proteins, and the
medium shows negligible leakage of the Ni2+ ion.
Ni Sepharose High Performance is compatible with all commonly used aqueous buffers, reducing
agents, and denaturants such as 6 M Gua-HCl and 8 M urea, as well as a range of other additives (see
Appendix 1). It is stable over a broad pH range. This high chemical and physical stability and broad
compatibility maintains the biological activity and increases the yield of the purified product, at the
same time as it greatly expands the range of suitable operating conditions, including procedures used
to clean the medium.
The good flow rates and distinctly separated peaks containing concentrated material make Ni Sepharose
High Performance the medium of choice for high-performance purifications. See Appendix 1 for the
main characteristics of Ni Sepharose High Performance.
Ni Sepharose High Performance is supplied preswollen in 20% ethanol, in pack sizes of 25 and 100 ml,
as well as in the convenient prepacked formats described later in this chapter.
Fig 11. Ni Sepharose High Performance precharged with Ni2+ for high-performance purification of histidine-tagged proteins.
Column packing
Refer to Appendix 4 for general guidelines for column packing.
Ideally, Sepharose High Performance media are packed in XK or Tricorn™ columns in a two-step procedure:
Do not exceed 1.0 bar (0.1 MPa) in the first step and 3.5 bar (0.35 MPa) in the second step. If the packing
equipment does not include a pressure gauge, use a packing flow rate of 5 ml/min (XK 16/20 column)
or 2 ml/min (Tricorn 10/100 column) in the first step, and 9 ml/min (XK 16/20 column) or 3.6 ml/min
(Tricorn 10/100 column) in the second step. If the recommended pressure or flow rate cannot be
obtained, use the maximum flow rate your pump can deliver. This should also give a well-packed bed.
1. Assemble the column (and packing reservoir if necessary).
2. Remove air from the end-piece and adapter by flushing with distilled water. Make sure no air has been
trapped under the column bed support. Close the column outlet leaving the bed support covered with
water.
3. Resuspend the medium and pour the slurry into the column in a single continuous motion. Pouring the
slurry down a glass rod held against the column wall will minimize the introduction of air bubbles.
4. If using a packing reservoir, immediately fill the remainder of the column and reservoir with water. Mount
the adapter or lid of the packing reservoir and connect the column to a pump. Avoid trapping air bubbles
under the adapter or in the inlet tubing.
5. Open the bottom outlet of the column and set the pump to run at the desired flow rate.
6. Maintain packing flow rate for at least 3 bed volumes after a constant bed height is reached. Mark the
bed height on the column.
7. Stop the pump and close the column outlet.
8. If using a packing reservoir, disconnect the reservoir and fit the adapter to the column.
9. With the adapter inlet disconnected, push the adapter down into the column until it reaches the mark.
Allow the packing solution to flush the adapter inlet. Lock the adapter in position.
10. Connect the column to a pump or a chromatography system and start equilibration. Readjust the
adapter if necessary.
Note: For subsequent chromatography procedures, do not exceed 75% of the packing flow rate.
Sample preparation
Refer to page 26 for a general procedure for sample preparation.
Adjust the sample to the composition and pH of the binding buffer by: adding buffer, NaCl,
imidazole, and additives from concentrated stock solutions; by diluting the sample with binding
buffer; or by buffer exchange. To prevent the binding of host cell proteins with exposed histidine,
it is essential to include imidazole at a low concentration in the sample and binding buffer (see
Chapter 4).
Pass the sample through a 0.22 µm or a 0.45 µm filter and/or centrifuge it immediately before
applying it to the column. If the sample is too viscous, to prevent it from clogging the column
dilute it with binding buffer, increase lysis treatment (sonication, homogenization), or add
DNase/RNase to reduce the size of nucleic acid fragments.
Buffer preparation
Binding buffer: 20 mM sodium phosphate, 0.5 M NaCl, 20 to 40 mM imidazole, pH 7.4 . (The optimal
imidazole concentration is protein dependent; 20 to 40 mM is suitable for many proteins.)
Elution buffer: 20 mM sodium phosphate, 0.5 M NaCl, 500 mM imidazole, pH 7.4
Water and chemicals used for buffer preparation should be of high purity. Filter buffers through a
0.45-µm filter before use. Use high-purity imidazole, as this will give a very low or no absorbance
at 280 nm.
The optimal concentration of imidazole needed in the sample and buffer to obtain the best
purity and yield differs from protein to protein. In the binding buffer, 20 to 40 mM imidazole is
suitable for many proteins; 500 mM imidazole in the elution buffer ensures complete elution of
the target protein.
As an alternative to elution with imidazole, lower the pH to approximately pH 4.5. (Metal ions will
be stripped off the medium below pH 4.0).
Purification
1. If the column contains 20% ethanol, wash it with 5 column volumes of distilled water. Use a linear flow
rate of 50 to 100 cm/h. Refer to Appendix 6 for flow rate calculations.
2. Equilibrate the column with 5 to 10 column volumes of binding buffer at a linear flow rate of 150 cm/h.
3. Apply the pretreated sample.
4. Wash with binding buffer until the absorbance reaches the baseline.
5. Elute with elution buffer using a step or linear gradient.
For step elution, 5 column volumes of elution buffer are usually sufficient.
For linear gradient elution, a shallow gradient, over 20 column volumes, may separate proteins with
similar binding strengths.
6. After elution, regenerate the column by washing it with 5–10 column volumes of binding buffer. The
column is now ready for a new purification.
The column does not need to be stripped and recharged between each purification if the same
protein is going to be purified. Reuse of any purification column depends on the nature of the
sample and should only be performed with identical proteins to prevent cross-contamination.
For more information on this topic and on cleaning and storage, refer to Appendix 1.
Use the elution buffer as blank when measuring absorbance manually. If imidazole needs to be
removed from the protein, use HiTrap Desalting, PD-10 Desalting, or HiPrep 26/10 Desalting
column.
Ni Sepharose is compatible with reducing agents. However, we recommend removal of any

weakly bound Ni2+ ions before applying buffer/sample that includes reducing agents. This can
be accomplished by performing a blank run without reducing agents (see below). Do not store
Ni Sepharose High Performance with buffers that include reducing agents.
Leakage of Ni2+ from Ni Sepharose is low under all normal conditions. The leakage is lower than
for other precharged IMAC media tested.
For very critical applications, leakage during purification can be even further diminished by
performing a blank run (as described below) before loading sample.
Blank run:
Use binding buffer and elution buffer without reducing agents.
1. Wash the column with 5 column volumes of distilled water (to remove the 20% ethanol).
2. Wash with 5 column volumes of elution buffer.
3. Equilibrate with 10 column volumes of binding buffer.
Purification using Ni Sepharose 6 Fast Flow
Ni Sepharose 6 Fast Flow consists of 90-µm beads of highly cross-linked agarose, to which a chelating
ligand has been immobilized and subsequently charged with Ni2+ ions. The ligand density of Ni Sepharose
6 Fast Flow ensures high binding capacity, and the medium shows negligible leakage of Ni2+ ions. The
high flow rate property of the Sepharose 6 Fast Flow matrix makes it well-suited for scaling-up but
also for gravity-flow purposes. In addition, the medium is compatible with a wide range of additives
commonly used in the purification of histidine-tagged proteins. See Appendix 1 for the main characteristics
of Ni Sepharose 6 Fast Flow.
Fig 12. Ni Sepharose 6 Fast Flow is designed for scaling up purification of histidine-tagged proteins but it works well also for gravity-
flow purification.
Ni Sepharose 6 Fast Flow is useful for batch/gravity-flow purification of histidine-tagged proteins using
Disposable PD-10 Columns. Ni Sepharose 6 Fast Flow prepacked in Disposable PD-10 Columns shows
excellent performance in terms of fast purification time and total protein recovered during gravity-flow
purification. See His GraviTrap on page 66 and also Data File 11-0008-86.
Ni Sepharose 6 Fast Flow is supplied preswollen in 20% ethanol, in pack sizes of 5, 25, 100, and 500 ml,
as well as in convenient prepacked formats as described later in this chapter.
Column packing
Ideally, Sepharose 6 Fast Flow media are packed in XK or Tricorn columns in a two-step procedure:
Do not exceed 0.5 bar (0.05 MPa) in the first step and 1.5 bar (0.15 MPa) in the second step. If the packing
equipment does not include a pressure gauge, use a packing flow rate of 2.5 ml/min (XK 16/20 column)
or 0.9 ml/min (Tricorn 10/100 column) in the first step, and 8.7 ml/min (XK 16/20 column) or 4.7 ml/min
(Tricorn 10/100 column) in the second step.
water.
Sample preparation
Chapter 4).
Buffer preparation
Water and chemicals used for buffer preparation should be of high purity. Filter buffers through a
0.45-µm filter before use. Use high-purity imidazole, as this will give a very low or no absorbance
at 280 nm.
the target protein.
be stripped off the medium below pH 4.0.)
Purification using a packed column
1. If the column contains 20% ethanol, wash it with 5 column volumes of distilled water. Use a linear flow
rate of 50 to 100 cm/h.
4. Wash with binding buffer until the absorbance reaches the baseline.
6. After elution, regenerate the column by washing it with 5–10 column volumes of binding buffer. The
protein is going to be purified. Reuse of any purification column depends on the nature of the sample
and should only be performed with identical tagged proteins to prevent cross-contamination.
removed from the protein, use HiTrap Desalting, a PD-10 Desalting, or HiPrep 26/10 Desalting
column.

Ni Sepharose 6 Fast Flow with buffers that include reducing agents.
Blank run:
Purification using batch/gravity-flow
Sample preparation
buffer; or by buffer exchange. To prevent the binding of host cell proteins with exposed
histidines, it is essential to include imidazole at a low concentration in the sample and binding
buffer (see Chapter 4).
Pass the sample through a 0.45 µm filter or centrifuge it immediately before applying it to the
column. If the sample is too viscous, to prevent it from clogging the column dilute it with binding
buffer, increase lysis treatment (sonication, homogenization), or add DNase/RNase to reduce the
size of nucleic acid fragments.
Buffer preparation
Water and chemicals used for buffer preparation should be of high purity. Use high-purity
imidazole, as this will give a very low or no absorbance at 280 nm.
the target protein.
Preparing the empty Disposable PD-10 Column

1. Wash the filter with 20% ethanol.
2. Rinse the filter with distilled water.
3. Insert the filter into the empty Disposable PD-10 Column. (Other empty gravity-flow columns can also be
used.)
Medium preparation
1. Gently shake the bottle until the slurry is homogeneous.
2. Remove a sufficient amount of slurry from the bottle and transfer to a centrifuge tube.
3. Sediment the Ni Sepharose 6 Fast Flow by centrifugation at 500 × g for 5 min.
4. Discard the supernatant and replace with 5 ml of distilled water.
5. Gently shake the slurry for 3 min and resediment by centrifugation at 500 × g for 5 min.
6. Repeat steps 4 and 5 using binding buffer instead of distilled water.
7. Transfer the slurry to a measuring cylinder.
8. Add an appropriate volume of binding buffer to make a 50% slurry.
Purification using gravity flow
1. Add sample to the 50% slurry. Binding capacity of Ni Sepharose 6 Fast Flow is protein dependent and the
average is 40 mg/ml. This means that 1 ml of the 50% slurry can bind approximately 20 mg of histidine-
tagged protein.
2. Incubate sample and the Ni Sepharose 6 Fast Flow slurry on a shaker at low speed for 1 h.
3. Load sample/Ni Sepharose 6 Fast Flow mix onto the PD-10 column and collect the flowthrough.
4. Wash with 2 to 5 medium volumes of binding buffer and collect the flowthrough. For example, if 0.5 ml of
Ni Sepharose 6 Fast Flow is used (1 ml of 50% slurry), wash with 1 to 2.5 ml of binding buffer.
5. Elute with 4 medium volumes of elution buffer and collect the eluted fractions in four separate tubes.
6. Measure absorbance at 280 nm using a spectrophotometer and confirm purity of the pooled fractions by
SDS-PAGE. Use elution buffer as the blank.

Ni Sepharose 6 Fast Flow with buffers that include reducing agents.
Blank run:
1. Wash the medium with 5 column volumes of distilled water (to remove the 20% ethanol).
2. Wash with 5 medium volumes of elution buffer.
3. Equilibrate with 10 medium volumes of binding buffer.
This can be done by centrifugal washes of the suspended medium or, much more efficiently, by
washing the medium on a sintered glass filter (medium grade G3 type).
High-throughput screening using His MultiTrap HP and
His MultiTrap FF 96-well filter plates
His MultiTrap HP and His MultiTrap FF are prepacked, disposable 96-well filter plates for reproducible,
high-throughput screening of histidine-tagged recombinant protein expression. Typical applications
are expression screening of different constructs, screening for solubility of proteins, and optimization
of the conditions for small-scale parallel purification. The plates are prepacked with precharged
Ni Sepharose High Performance and Ni Sepharose 6 Fast Flow, respectively.
Each well of the prepacked His MultiTrap HP and His MultiTrap FF contains 500 µl of a 10% slurry of
Ni Sepharose High Performance or Ni Sepharose 6 Fast Flow in storage solution (50 µl of medium in
20% ethanol) and has a capacity for purifying up to 1.0 mg and 0.8 mg of histidine-tagged protein,
respectively. The plates are made of polypropylene and polyethylene.
Characteristics of the media and of His MultiTrap HP and His MultiTrap FF are listed in Appendix 1.
The Ni2+-charged media are compatible with all commonly used aqueous buffers, reducing agents,
denaturants, such as 6 M Gua-HCl and 8 M urea, and a range of other additives.
Prepacked His MultiTrap HP and His MultiTrap FF plates provide well-to-well and plate-to-plate
reproducibility in terms of yield and purity of eluted protein. Automated robotic systems can be used,
as well as manual handling using centrifugation or vacuum pressure. The purification procedure can
easily be scaled up because Ni Sepharose is available in both larger prepacked formats and as lab
packs. Scaling up from His MultiTrap plates to a HisTrap 1-ml or 5-ml column while keeping the same
conditions (e.g., Fast Flow or High Performance medium, imidazole concentration, etc.) provides highly
consistent results and shortens the optimization time at scale-up.
Fig 13. His MultiTrap HP and His MultiTrap FF are prepacked 96-well filter plates for high-throughput expression screening of histidine-
tagged proteins.
Sample preparation
Lysis with commercial kits could give large cell debris particles that may interfere with drainage
of the wells during purification. This problem can be solved by centrifugation or filtration of the
sample before adding it to the wells.
After thorough cell disruption, it is possible to apply unclarified lysate directly to the wells
without pre-centrifugation and/or filtration of the sample.
Apply the unclarified lysate to the wells directly after preparation, as the lysate may precipitate
unless used immediately or frozen before use. New lysing of the sample can then prevent
clogging of the wells when loading the plate.
If the sample is too viscous, an extension of the duration of mechanical treatment of the sample
to ensure a more complete lysis is recommended (keep the sample on ice to prevent overheating).
Buffer preparation
Binding buffer: 20 mM sodium phosphate, 500 mM NaCl, 20 to 40 mM imidazole, pH 7.4. (The optimal
Elution buffer: 20 mM sodium phosphate, 500 mM NaCl, 500 mM imidazole, pH 7.4.
To increase the purity, use as high a concentration of imidazole as possible in the sample and
binding buffers without losing binding capacity. Refer to Chapter 4 for additional information on
this topic.
Centrifugation procedure for high-throughput screening

Preparing the filter plate
1. Peel off the bottom seal from the 96-well filter plate. Be sure to hold the filter plate over a sink to
accommodate any leakage of storage solution when removing the bottom seal.
2. Hold the filter plate upside down and gently shake it to dislodge any medium adhering to the top seal.
Return the filter plate to an upright position.
3. Place the filter plate against the bench surface and peel off the top seal.
4. Position the filter plate on top of a collection plate.
Note: Remember to change or empty the collection plate as necessary during the following steps.
5. Centrifuge the filter plate for 2 min at 500 × g to remove the ethanol storage solution from the medium.
6. Add 500 µl of deionized water to each well. Centrifuge the plate for 2 min at 500 × g.
7. Add 500 µl of binding buffer to each well to equilibrate the medium. Centrifuge for 2 min at 500 × g.
Repeat once. The filter plate is now ready for use.
Blank run: Reducing agents may be used in sample and buffers. In such a case, perform a blank
run by applying 500 µl of elution buffer/well before step 7. No reducing agent should be used
in buffer during blank runs. Reequilibrate with binding buffer including reducing agent before
sample application. Do not leave His MultiTrap plates with buffers including reducing agents
when not in use.
Centrifugation procedure
Do not apply a force of more than 700 × g during centrifugation.
1. Apply unclarified or clarified lysate (maximum 600 µl per well) to the wells of the filter plate and incubate
for 3 min.
Note: If the yield of protein is too low, increase the incubation time and/or gently agitate the filter plate to
effect mixing.
2. Centrifuge the plate at 100 × g for 4 min or until all the wells are empty. Discard the flowthrough.
3. Add 500 µl of binding buffer per well to wash out any unbound sample. Centrifuge at 500 × g for 2 min.
Repeat once or until all unbound sample is removed.
4. Add 200 µl of elution buffer per well and mix for 1 min.
Note: The volume of elution buffer can be varied (50 to 100 µl per well), depending on the concentration of
target protein required.
5. Change the collection plate and centrifuge at 500 × g for 2 min to collect the eluted protein. Repeat twice
or until all the target protein has been eluted.
Note: High-purity protein should yield an A280 reading of < 0.1. If necessary, change the collection plate
between each elution to prevent unnecessary dilution of the target protein.
Vacuum procedure for high-throughput screening
If problems with foaming, reproducibility, or bubbles in the collection plate occur using vacuum,
the centrifugation procedure should be considered. The distance between the filter plate and
the collection plate is critical; adjust the distance if necessary.

5. Set the vacuum to -0.15 bar. Place the 96-well plate and collection plate on the vacuum manifold to
remove the ethanol storage solution from the medium.
6. Add 500 µl of deionized water to each well. Apply vacuum to drain the water from the wells.
7. Add 500 µl of binding buffer to each well to equilibrate the medium. Remove the solution as in step 5.
Repeat once. The filter plate is now ready for use.
Blank run: Reducing agents may be used in sample and buffers. In such a case, run a blank run
by applying 500 µl of elution buffer/well before step 7. No reducing agent should be used in
buffer during blank runs. Reequilibrate with binding buffer including reducing agent before
sample application. Do not leave His MultiTrap plates with buffers including reducing agents
when not in use.
Vacuum procedure
Do not apply a pressure in excess of -0.5 bar during vacuum operation.
If a robotic system is used for purification, the vacuum must be adjusted according to methods
applicable to the system.
for 3 min.
Note: If the yield of protein is too low, increase the incubation time and/or gently agitate the filter plate.
2. Remove the flowthrough by applying a vacuum of -0.15 bar until all the wells are empty. Slowly increase
the vacuum to -0.30 bar and turn off the vacuum after approximately 5 sec. Discard the flowthrough.
Increasing the vacuum too quickly can result in foaming under the filter plate and subsequent
cross-contamination of samples.
3. Add 500 µl of binding buffer per well to wash out any unbound sample. Apply a vacuum of -0.15 bar as in
step 2. Repeat once or until all unbound sample is removed.
Note: The volume of elution buffer can be varied (50 to 100 µl per well), depending on the concentration of
target protein required.
5. Change the collection plate and apply a vacuum of -0.15 bar to collect the eluted protein. Repeat twice or
until all the target protein has been eluted.
Note: High-purity protein should yield an A280 reading of < 0.1. If necessary, change the collection plate
between each elution to prevent unnecessary dilution of the target protein.
Application example
Determining solubility effects of detergents in buffers during purification of membrane
proteins using His MultiTrap FF
The 96-well plate format of His MultiTrap FF and His MultiTrap HP allows high-throughput screening
and purification of histidine-tagged proteins. In this example, His MultiTrap FF was used to screen eight
detergents for their effect on the solubility of six histidine-tagged membrane proteins. Results from
purification screening of two proteins, GlpG protein (EM29) and cation transporter (EM43), are shown in
a dot blot and SDS-PAGE in Figure 14. The results show that conditions to find the most appropriate
detergent for the membrane proteins in the study can be readily optimized, with high reproducibility,
using MultiTrap 96-well filter plate.
96-well filter plate:
His MultiTrap FF
Sample: Six E. coli lysates containing histidine-tagged membrane proteins: probable transporter, ion transporter,
putative transferase, regulatory protein, GlpG protein, and cation transporter; GlpG protein (EM29)
and cation transporter (EM43) are shown here
Sample preparation: Chemical and freeze/thaw lysis
Sample volume: 100 µl/well
Elution method: Centrifugation
Elution volume: 3 × 50 µl/well
Lysis buffer: 20 mM sodium phosphate, pH 7.4, 100 mM sodium chloride, 20 mM imidazole, 0.5 mM TCEP,
5 U/ml Benzonase™ Nuclease, 1 mg/ml lysozyme, EDTA-free protease inhibitor cocktail,
1% to 2% detergent , and 1X BugBuster™ Protein
Extraction Reagent , 25 U/ml Benzonase Nuclease, 1 kU/ml rLysozyme™ Solution, and 2X Complete Protease
Inhibitor Cocktail Tablet solution
Binding buffer: 20 mM sodium phosphate, pH 7.4, 500 mM sodium chloride, 20 mM imidazole, 0.5 mM TCEP, 1–2% detergent
Wash buffer: 20 mM sodium phosphate, pH 7.4, 500 mM sodium chloride, 40 mM imidazole, 0.5 mM TCEP, 0.03% DDM,
1–2% detergent
Elution buffer: 20 mM sodium phosphate pH 7.4, 500 mM sodium chloride, 500 mM imidazole, 0.5 mM TCEP, 0.03% DDM,
1–2% detergent
Detergents: 1% Fos-Choline 12 (FC12), 1% undecyl maltoside (UDM), 1% dodecyl maltoside (DDM), 1% Cymal-5,
1% Cymal-6, 2% octyl glucoside (OG), 1% Triton™ X-100 (TX-100), 1% lauryl dimethylamine oxide (LDAO)
Data evaluation: Dot-blot analysis on nitrocellulose membrane. Histidine-tagged proteins were detected using
HisProbe™-HRP chemistry. SDS-PAGE with Coomassie staining
A) B) FC12 DDM Cymal 6 TX-100 FC12 DDM Cymal 6 TX-100

Standard protein
EM29/Elution 1
EM29/Elution 1
EM29/Elution 2
EM29/Elution 2
EM29/Elution 3
EM43/Elution 1
EM43/Elution 1
EM43/Elution 2
EM43/Elution 2
EM43/Elution 3
UDM Cymal 5 OG LDAO UDM Cymal 5 OG LDAO
Mr
FC12 0.53 mg/ml 97 000
UDM 0.053 mg/ml 66 000
DDM 0.027 mg/ml 45 000
Cymal 5 0.013 mg/ml 30 000
Cymal 6 20 100
OG
TX-100 14 400
LDAO
Fig 14. (A) Dot blot of membrane proteins EM29 and EM43 purified on His MultiTrap FF in the presence of different detergents. Repeats of
eluates 1 and 2 shown in the dot blot are two independent extractions and purifications. (B) SDS-PAGE (Coomassie staining) of EM29
purifications (elutions 1 and 2 in the blot) with eight different detergents on His MultiTrap FF.
Minipreps using His SpinTrap
His SpinTrap is designed for efficient minipreps (small-scale purification) of histidine-tagged proteins
directly from clarified or unclarified cell lysates. The columns may also be used for screening of large
numbers of small-scale lysates, as well as optimization of purification conditions. The columns are
prepacked with Ni Sepharose High Performance. Refer to Appendix 1 for the main characteristics of
His SpinTrap.
1 2 3 4
Fig 15. His SpinTrap is a single-use column for simple, small-scale purification of histidine-tagged proteins and rapid expression screening.
Purifying histidine-tagged proteins with His SpinTrap is a simple, four-stage procedure that can be performed in 10 min using a
microcentrifuge: (1) After placing the column in a 2-ml microcentrifuge tube, equilibrate by adding binding buffer and centrifuge;
(2) add sample; (3) wash with binding buffer; (4) elute the target protein with elution buffer.
His SpinTrap columns are used together with a standard microcentrifuge. One purification run takes
approximately 10 min. For optimal performance, use His SpinTrap together with buffers prepared using
His Buffer Kit. Purification of unclarified samples on His SpinTrap columns minimizes loss of target
protein caused by manual operations such as sample precentrifugation, transfer to centrifugation
tubes, and collecting supernatant. In addition, loading unclarified sample directly to the His SpinTrap
columns reduces sample preparation time, which minimizes degradation of sensitive target proteins.
See Figures 15 and 16 for schematics showing the procedure.
Unclarified sample
Enzymatic His SpinTrap column
and purification
mechanical
cell lysis
approx.
40 min 10 min 50 min
Clarified sample
Enzymatic His SpinTrap column
and Transfer purification
mechanical to sample Collect
cell lysis tubes Centrifugation supernatant
approx.
30–60 min 80–110 min
40 min 10 min
Fig 16. Total times for preparing and purifying unclarified samples are 30 to 60 min less than for clarified samples because the extra
time needed to clarify the cell lysate by centrifugation is eliminated.
Sample preparation
The procedure below has been used successfully in our own laboratories for sample preparation
prior to use of His SpinTrap, but other established procedures may also work. Use standard 2-ml
microcentrifuge tubes.
1. Dilute the cell paste: Add 1 ml of binding buffer to resuspend cell paste obtained from 20 to 50 ml of cell
culture (depending on expression level).
To prevent host cell proteins binding to exposed histidines, it is essential that the sample and
binding buffers contain the same concentration of imidazole.
2a. Enzymatic lysis: Add 0.2 mg/ml lysozyme, 20 µg/ml DNase, 1 mM MgCl2, and 1 mM Pefabloc SC or PMSF.
Vortex the tubes gently and incubate at room temperature for 30 min.
Chemical lysis kits can also be used, but make sure that they do not contain any chelating agent.
2b. Mechanical lysis: Disrupt cells by repeated freeze/thaw, homogenization, or sonication.
You can also apply clarified sample to the column by spinning at full speed in a microcentrifuge
for 10 min to remove insoluble material. Collect supernatant and purify on His SpinTrap.
Buffer preparation
Recommended buffers for native conditions can easily be prepared from His Buffer Kit.
Native conditions:
Binding buffer: 20 mM sodium phosphate, 500 mM NaCl, 20 mM imidazole, pH 7.4
Elution buffer: 20 mM sodium phosphate, 500 mM NaCl, 500 mM imidazole, pH 7.4
Denaturing conditions:
Binding buffer: 20 mM Tris-HCl, 8 M urea, 500 mM NaCl, 5 mM imidazole, pH 8.0 + 1 to 5 mM β-mercapto-
ethanol
Elution buffer: 20 mM Tris-HCl, 8 M urea, 500 mM NaCl, 500 mM imidazole, pH 8.0 + 1 to 5 mM β-mercapto-
ethanol
purity and yield differs from protein to protein. Under native conditions, 20 to 40 mM imidazole
in the binding buffer is suitable for many proteins; 500 mM imidazole in the elution buffer is
most often sufficient to completely elute the target protein.
As an alternative to elution with imidazole, you can lower the pH to approximately pH 4.5. (Note
that metal ions will be stripped off the medium below pH 4.0.)
Purification
Perform purifications on His SpinTrap using a standard microcentrifuge. Place the column in a 2-ml
microcentrifuge tube to collect the liquid during centrifugation. Use a new 2-ml tube for every step
(steps 1 to 6).
1. Invert and shake the column repeatedly to resuspend the medium. Loosen the top cap one-quarter of a
turn and break off the bottom closure.
2. Place the column in a 2-ml microcentrifuge tube and centrifuge for 30 s at 70 to 100 × g (approx.
1000 rpm in an Eppendorf™ 5415R, 24-position fixed-angle rotor) to remove the storage liquid.
3. Remove and discard the top cap. Equilibrate the column by adding 600 µl of binding buffer. Centrifuge for
30 s at 70 to 100 × g.
4. Add up to 600 µl (total) of prepared sample. Centrifuge for 30 s at 70 to 100 × g.
You can make several sample applications as long as you do not exceed the binding capacity of
the column (see Appendix 1).
5. Wash with 600 µl of binding buffer. Centrifuge for 30 s at 70 to 100 × g.
6. Elute the target protein twice with 200 µl of elution buffer. Centrifuge for 30 s at 70 to 100 × g and collect
the purified sample. The first 200 µl will contain the majority of the target protein.
Application example
Purification of unclarified sample using His SpinTrap
The performance of His SpinTrap columns in purifying a histidine-tagged protein from unclarified E. coli
lysate was assessed. Histidine-tagged green fluorescent protein, GFP-(His)6, in E. coli BL-21 lysate, was
subjected to enzymatic lysis followed by sonication for 10 min, and the unclarified lysate was loaded
directly on His SpinTrap. For comparison, half of the sample was also clarified by centrifugation before
purification. Samples and binding buffer contained 60 mM imidazole. To ensure complete elution of
GFP-(His)6, which has a high affinity for Ni Sepharose High Performance, the elution buffer contained
800 mM imidazole rather than the more usual 500 mM. Purification time for the unclarified and clarified
sample was 10 min. The final purity of eluates from unclarified and clarified samples was similar as
confirmed by SDS-PAGE (Fig 17).
Column: His SpinTrap
Equilibration: 600 µl binding buffer
Mr Sample application: 600 µl unclarified or clarified E. coli
97 000 BL-21 lysate containing 150 µg GFP-(His)6
Wash: 600 µl binding buffer
66 000 Elution: 2 × 200 µl elution buffer
Binding buffer: 20 mM sodium phosphate, 500 mM NaCl,
45 000 60 mM imidazole, pH 7.4
30 000 Elution buffer: 20 mM sodium phosphate, 500 mM NaCl,
800 mM imidazole, pH 7.4
20 100
Lanes
14 400
1. LMW markers
2. Unclarified sample, start material (diluted 1:10)
3. Clarified sample, start material (diluted 1:10)
4. Unclarified sample, eluted pool
1 2 3 4 5 5. Clarified sample, eluted pool
Fig 17. SDS-PAGE (ExcelGel™ SDS Gradient 8–18) under reducing conditions of unclarified and clarified E. coli lysate containing
GFP-(His)6. Similar purity and recovery were observed for both unclarified and clarified sample.
Purification using HisTrap HP and HisTrap FF
HisTrap HP and HisTrap FF are 1-ml and 5-ml HiTrap columns packed with Ni Sepharose High Performance
or Ni Sepharose 6 Fast Flow, respectively. Sample application, washing, and elution can be performed
using a syringe with a supplied adapter, a peristaltic pump, or a liquid chromatography system such
as ÄKTAdesign (see Table 8 for equipment choices).
HisTrap HP and HisTrap FF columns are made of polypropylene, which is biocompatible and non-
interactive with biomolecules. The top and bottom frits are manufactured from porous polyethylene.
Columns are delivered with a stopper on the inlet and a snap-off end on the outlet. Every package
includes all necessary components for connection of the columns to different types of equipment.
For quick scale-up of purifications, two or three HisTrap columns (1 ml or 5 ml) can be connected in
series (back pressure will be higher). Note that HisTrap HP and HisTrap FF columns cannot be opened
or refilled.
Equilibrate column Apply sample Elute

with wash with with
binding buffer binding buffer elution buffer
3 min 5 to 15 min 2 min
Waste Collect flowthrough Collect fractions
Fig 18. HisTrap HP and HisTrap FF 1-ml and 5-ml columns allow convenient and simple one-step purification of histidine-tagged
proteins. HisTrap HP 1-ml and 5-ml columns shown here. The simple purification scheme is shown at right.
Sample preparation
buffer; or by buffer exchange. To prevent the binding of host cell proteins with exposed histidines,
Chapter 4).
Buffer preparation
a 0.22 µm or a 0.45 µm filter before use. Use high-purity imidazole as this will give very low or
no absorbance at 280 nm.
As an alternative to elution with imidazole, you can lower the pH to approximately pH 4.5. (Note
that metal ions will be stripped off the medium below pH 4.0.)
Purification
1. Fill the syringe or pump tubing with distilled water. Remove the stopper and connect the column to the
syringe (use the connector supplied), laboratory pump, or chromatography system “drop to drop” to
avoid introducing air into the system.
2. Remove the snap-off end at the column outlet.
3. Wash out the ethanol with 3 to 5 column volumes of distilled water.
4. Equilibrate the column with at least 5 column volumes of binding buffer. Recommended flow rates are
1 ml/min (1-ml column) and 5 ml/min (5-ml column).
5. Apply the pretreated sample using a syringe fitted to the Luer connector or by pumping it onto the column.
For best results, use a flow rate of 0.2 to 1 ml/min (1-ml column) and 0.5 to 5 ml/min (5-ml column) during
sample application*.
6. Wash with binding buffer (generally at least 5 to 10 column volumes) until the absorbance reaches a
steady baseline or no material remains in the effluent. Maintain a flow rate of 1 to 2 ml/min (1-ml column)
and 5 to 10 ml/min (5-ml column) for washing.
7. Elute with elution buffer using a one-step or linear gradient. For step elution, 5 column volumes is usually
sufficient. For linear gradient elution, 10 to 20 column volumes are usually sufficient. Maintain a flow rate
of 1 to 2 ml/min (1-ml column) and 5 to 10 ml/min (5-ml column) for elution.
8. After elution, regenerate the column by washing it with 3 to 5 column volumes of binding buffer. The
*One ml/min corresponds to approximately 30 drops/min when using a syringe with a HiTrap 1-ml
column, and 5 ml/min corresponds to approximately 120 drops/min when using a HiTrap 5-ml column.

HisTrap columns with buffers that include reducing agents.
Blank run:
Application examples
1. One-step on-column refolding and purification of a histidine-tagged protein from
inclusion bodies using HisTrap HP
Histidine-tagged, solubilized single chain Fv antibody fragment Fab 57P, 10 ml (concentration 0.67 mg/ml)
from E. coli inclusion bodies was refolded and purified using a 1-ml HisTrap HP column. The yield from
refolding was 14% according to analysis using Biacore™ System 2000; see Figure 19B. The parameters
of refolding could be optimized and the final method automated for both refolding and purification
using HisTrap HP. Figure 19A-C shows the results from the refolding, purification, and analysis.
Sample: Histidine-tagged, solubilized single chain Fv antibody fragment Fab 57,

10 ml (conc. 0.67 mg/ml) E. coli inclusion bodies
Column: HisTrap HP 1 ml
Solubilizing buffer: 20 mM Tris-HCl, 6 M Gua-HCl, 1 mM DTE, 1 mM Na2-EDTA, 0.1 mM Pefabloc, pH 7.5
Denatured binding buffer: 20 mM Tris-HCl, 5 mM imidazole, 0.5 M NaCl, 8 M urea, 1 mM DTE, 0.1 mM Pefabloc, pH 7.5
Refolding buffer: 20 mM Tris-HCl, 5 mM imidazole, 0.5 M NaCl, 0.5 M arginine-HCl, 1 mM reduced glutathione (GSH),
1 mM oxidized glutathione (GSSG), pH 7.5
Native binding buffer: 20 mM Tris-HCl, 10 mM imidazole, 0.5 M NaCl, pH 7.5
Native elution buffer: 20 mM Tris-HCl, 500 mM imidazole, 0.5 M NaCl, pH 7.5
Flow rate: 1 ml/min
System: ÄKTAexplorer 10
A) B)
mAU RU
3 5 Elution buffer % 20000
100
500 15000
Resp.Diff.
80 10000
400
5000
60 0
300 1
-5000
200 400 600 800 1000 1200 1400 1600
200 40 Sec
Reference cell
4 Sample cell
2 Response difference when reference cell is subtracted
100 20
System: Biacore System 2000
0 Sensor surface: Immobilized peptide C16V”37” (tobacco
0
mosaic virus) with affinity to scFv57P
0 20 40 60 80 100 ml
Blank surface: Immobilized with peptide with no affinity to
scFv57P
1 Inject
Yield: 14 % active protein from pooled fraction
2 Washing the system first with native elution buffer and, second, washing
the system with native binding buffer. Note the column valve in bypass
mode. Third, start to equilibrate the column with native binding buffer.
3 Start refolding using refolding buffer
4 Recondition of the system to native conditions
5 Start elution using native elution buffer
Pooled fraction from purification of

refolded scFv 57P antibody fragment
C) Elution buffer %
mAU
100
500
80
400
300 60
200 40
100 20
pool
0
0
91.5 92 92.5 93 93.5 94 94.5 95 ml
Fig 19 (A). On-column folding and purification. (B) Sensogram of the interaction between immobilized peptide and refolded protein.
(C) Enlarged figure of pooled fraction from purification of refolded scFv 57P antibody fragment.
2. Two-step purification of a high-molecular-weight histidine-tagged protein
using HisTrap HP
The high-molecular-weight protein histidine-tagged mannanase Man 26A from Cellulomonas fimi
(Mr 100 000) was purified in its enzymatically active form using a 1-ml HisTrap HP column (Fig 20A).
A second purification step using gel filtration with Superdex™ 200 was added to obtain a purity of 95%
(Figs 20B and 20C, respectively).
A. Affinity chromatography (AC) B. Gel filtration (GF)
Sample: 10 ml E. coli extract with low-level expression of Sample: 0.5 ml concentrated sample from
a histidine-tagged mannanase, Man 26A, from HisTrap HP 1-ml column
Cellulomonas fimi (Mr ~ 100 000) Column: Superdex 200 10/300 GL
Column: HisTrap HP 1 ml Buffer: PBS, pH 7.5
Binding buffer: 20 mM sodium phosphate, 30 mM imidazole, Flow rate: 0.5 ml/min
500 mM NaCl, pH 7.4 System: ÄKTAexplorer 100
Elution buffer: 20 mM sodium phosphate, 500 mM imidazole,
500 mM NaCl, pH 7.4
Gradient: 25 ml linear gradient 30–300 mM imidazole
Flow rate: 1 ml/min
A) B)
A 280 mAU
mAU 80.0
400 AC 70.0 GF
60.0
300 50.0
40.0
200
30.0
20.0
100
10.0
0 Eluted pool 0.0
0.0 10.0 20.0 30.0 40.0 50.0 ml 0.0 5.0 10.0 15.0 20.0 25.0 30.0 ml
C)
Mr
97 000
66 000
45 000
Lanes
1. LMW markers 30 000
2. E. coli extract , start material
3. HisTrap HP flowthrough 20 100
4. Early elution fraction, HisTrap HP fraction
14 400
5. HisTrap HP, eluted pool
6. Superdex 200 10/300 GL, pool 1 2 3 4 5 6
Fig 20. (A) First purification step, affinity chromatography, using HisTrap HP 1-ml column. (B) Second purification step with gel filtration
using Superdex 200 30/100 GL. (C) SDS-PAGE.
3. Scaling up purification from HisTrap FF to pilot scale
Histidine-tagged maltose binding protein MBP-(His)6 was purified from an E. coli extract. Samples
containing 8, 40, and 160 mg, respectively, were loaded on a 1-ml HisTrap FF column, a 5-ml HisTrap
FF column, and a 20-ml HisPrep FF 16/10 column, all of which were run at the same linear flow rate.
The results show that scaling up the column dimension while running at the same linear flow rate
provides highly consistent results (Fig 21A–C). Pooled fractions were analyzed by SDS-PAGE and
showed almost identical results in terms of purity and recovery (Fig 21D).
To go from laboratory to pilot scale, higher sample load is necessary. Scale-up was conducted with a
high load (88% of the binding capacity) of MBP-(His)6. The high sample load required optimization of
the binding and washing buffer to avoid loss of MBP-(His)6 during the washing step. An imidazole
concentration of 5 mM was found to give the best recovery and purity results. Two separate runs were
conducted using HisPrep FF 16/10 columns to show the reproducibility of the purification. The protocol
was then scaled up 10-fold. Pooled fractions analyzed by SDS-PAGE gave almost identical results in
terms of recovery and purity between the different runs and different scales to indicate a successful
process scale-up (Figure 22B).
A) Columns: HisTrap FF 1 ml, HisTrap FF 5 ml,
A 280 HisTrap FF, 1-ml HisPrep FF 16/10 (20 ml)
mAU Sample: Histidine-tagged Maltose Binding Protein,
4000 MBP-(His)6, in E. coli extract
3500 Binding buffer: 20 mM sodium phosphate, 25 mM imidazole,
500 mM NaCl, pH 7.4
3000
Elution buffer: 20 mM sodium phosphate, 500 mM imidazole,
2500
500 mM NaCl, pH 7.4
2000 Flow rates: HisTrap FF 1 ml: 1 ml/min
6.2 mg
1500 HisTrap FF 5 ml: 5 ml/min
1000 HisPrep FF 16/10: 5 ml/min
500
0
0.0 5.0 10.0 15.0 20.0 25.0 30.0 40.0 ml
D)
B)
A 280 HisTrap FF, 5-ml
mAU
4000
3500
3000 33 mg Mr
2500 97 000
2000
1500 66 000
1000 45 000
500
0 30 000
0.0 50 100 150 ml
20 100
C)
A 280 HisPrep FF 16/10, 20-ml 14 400
mAU
4000 1 2 3 4 5
3500 Lanes
3000 1. LMW markers
149 mg
2. Start material
2500
3. Eluted pool, HisTrap FF 1 ml
2000 4. Eluted pool, HisTrap FF 5 ml
1500 5. Eluted pool, HisPrep FF 16/10 (20 ml)
1000
500
0
0 100 200 300 400 500 600 700 ml
Fig 21. Scale-up from (A) HisTrap FF 1 ml via (B) HisTrap FF 5 ml to (C) a HisPrep 16/10 (20 ml) prepacked column. The samples loaded
contained approximately 8, 40, and 160 mg of MBP-(His)6, respectively. Recovery in milligrams is shown in each chromatogram.
(D) SDS-PAGE (ExcelGel SDS Gradient 8–18) under nonreducing conditions confirms that scaling up from the 1-ml to the 20-ml column
does not significantly affect the purification result.
A) C)
mAU HisPrep FF 16/10
3000 Mr
97 000
2500 66 000
2000 45 000
30 000
1500
20 100
1000 14 400
500
1 2 3 4 5 6 7 8 9 10 11 12
0
Lanes
0 100 200 300 400 ml 1. LMW markers
B) 2. Sample loaded, E. coli extract
AxiChrom 50 3. Flowthrough HisPrep FF 16/10 run 1
mAU
4. Wash HisPrep FF 16/10 run 1
5. Eluted pool HisPrep FF 16/10 run 1
4000 6. Flowthrough HisPrep FF 16/10 run 2
7. Wash HisPrep FF 16/10 run 2
3000 8. Eluted pool HisPrep FF 16/10 run 2
9. Flowthrough AxiChrom 50 run
2000 10. Wash AxiChrom 50 run
11. Eluted pool AxiChrom 50 run
1000 12. LMW markers
0
0 1000 2000 3000 4000 ml
Fig 22. Scale-up from (A) HisPrep FF 16/10 (20 ml) to (B) AxiChrom™ 50 (210 ml) column. ÄKTAexplorer 100 was used for the purification
runs on HisPrep FF 16/10 columns and ÄKTApilot was used for AxiChrom 50 purification. All systems were controlled by UNICORN
software. Note that only the chromatogram for run 1 on HisPrep FF 16/10 is presented. (C) SDS-PAGE of various fractions from both
purifications.
Purification using HisTrap FF with ÄKTAprime plus
These procedures use a HisTrap FF 1-ml column but also can be used with a HisTrap HP 1-ml
column.
Buffer preparation
Binding buffer (port A1): 20 mM sodium phosphate, 0.5 M NaCl, 20 mM imidazole, pH 7.4.
Elution buffer (port B): 20 mM sodium phosphate, 0.5 M NaCl, 0.5 M imidazole, pH 7.4.
Use high-purity water and chemicals and filter all buffers through a 0.45 µm filter before use.
Prepare at least 500 ml of eluent.
20 to 40 mM imidazole should be included in the binding buffer to reduce nonspecific binding of

non-histidine-tagged proteins. The concentration of imidazole is protein dependent, and if the
protein of interest elutes or does not bind at a certain imidazole concentration, then reduce the
concentration.
Sample preparation
Chapter 4).
System preparation
This example uses ÄKTAprime plus. Once the system is prepared, the remaining steps (under Selecting
Application Template and starting the method) will be performed automatically.
1. Place each inlet tubing from port A (8-port valve) in the binding buffer and the tubing from port B (2-port
valve) in the elution buffer.
2. Place the three brown waste tubings in waste.
3. Connect the column between port 1 on the injection valve (7-port valve) and the UV flow cell.
4. Fill the fraction collector rack with 18-mm tubes and position the white plate on the fractionation arm
against the first tube.
For step elution, the number of tubes to insert in the fraction collector will vary with the sample
volume. Fill the fraction collector with 20 tubes + one tube/ml sample. For example, if the
sample volume is 10 ml, fill the fraction collector with 20 + 10 = 30 tubes. However, note that the
maximum capacity of the fraction collector is 95 tubes, limiting the sample volume to 75 ml.
For gradient elution, use a minimum of 40 tubes.
5. Connect a sample loop large enough for your sample between port 2 and 6 on the injection valve. Use a
syringe to manually fill the loop.
Note: If a Superloop™ is needed, additional information is supplied in the instructions for Superloop.
Selecting Application Template and starting the method for step elution
1. Check the communication to PrimeView™. At the lower right corner of the screen the text Controlled By:
prime should be displayed.
2. Use the arrow and OK buttons to move in the menu tree until you find Affinity purification any HiTrap.
Set Sample Inj. Vol
Templates
(00.0 ml) 00.0
Application Template Run Application Template

Press OK to start
Affinity Purification
Run data displayed
any HiTrap
3. Enter the sample volume and press OK to start the template.
%B Elution
100
Priming
Equilibration Water wash

50 & priming
Sample
Reequili-
bration
1 10 20 10 5 Min
Fig 23. Theoretical gradient in Affinity Purification any HiTrap Application Template. Total separation time = 47 min + sample
application time.
AU280 %B Sample: Clarified homogenate of E. coli

100 expressing histidine-tagged protein
UV 280 nm Column: HisTrap HP 1 ml
2.5 Programmed %B Binding buffer (port A1): 20 mM phosphate, 0.5 M NaCl,
80 20 mM imidazole, pH 7.4
Elution buffer (port B): 20 mM phosphate, 0.5 M NaCl,
2.0
0.5 M imidazole, pH 7.4
60
1.5
40
1.0
20
0.5
0 0
5 10 15 20 25 30 35 min
Fig 24. Typical result from step elution of a histidine-tagged protein.
Selecting Application Template and starting the method for gradient elution
1. Check the communication to PrimeView. At the lower right corner of the screen the text Controlled By:
2. Use the arrow and OK buttons to move in the menu tree until you find His Tag Purification HisTrap.
Set Sample Inj. Vol
Templates
(00.0 ml) 00.0

Press OK to start
His Tag Purification

Run data displayed
HisTrap
%B Wash
100
Priming
Elution
Equilibration
50
Sample
Buffer wash
Reequili-
bration
2 10 20 20 17 5 Min
Fig 25. Theoretical gradient in His Tag Purification HisTrap Application Template. Total separation time = 74 min + sample application
time.
AU280 %B Sample: Clarified homogenate of E. coli

expressing histidine-tagged protein
100
UV 280 nm Column: HisTrap HP 1 ml
Programmed %B Binding buffer (port A1): 20 mM phosphate, 0.5 M NaCl,
1.5
Elution buffer (port B): 20 mM phosphate, 0.5 M NaCl,
0.5 M imidazole, pH 7.4
60
1.0
40
0.5
20
0 0
0 10 20 30 40 50 60 min
Fig 26. Typical result from gradient elution of a histidine-tagged protein.
Purification from unclarified cell lysate using
HisTrap FF crude
HisTrap FF crude is a ready-to-use column, prepacked with precharged Ni Sepharose 6 Fast Flow, for
purification of histidine-tagged recombinant proteins. After thorough cell disruption, it is possible to
load the unclarified, crude cell lysate directly on the column without centrifugation and filtration of
the sample.
Direct loading of unclarified cell lysates decreases the total purification time and may increase
the possibility of purifying sensitive target proteins without losing their activity.
Fig 27. HisTrap FF crude columns allow simple, one-step purification of histidine-tagged proteins without sample pretreatment such as
centrifugation and filtration.
HisTrap FF crude columns are made of polypropylene that is biocompatible and does not interact with
biomolecules. Columns are delivered with a stopper on the inlet and a snap-off end on the outlet. The
columns have porous polyethylene top and bottom frits with a pore size optimized for loading of
unclarified cell lysates directly on the column without causing back-pressure problems or leakage of
the Ni Sepharose 6 Fast Flow beads. Columns can be operated with a syringe and the supplied Luer
connector, a peristaltic pump, or a chromatography system such as ÄKTAdesign. Note that HisTrap FF
crude columns cannot be opened or refilled.
HisTrap FF crude
Enzymatic
and
mechanical
cell lysis Purification run
approx.
40 min 40 min 80 min
Conventional IMAC
Enzymatic
and Transfer
mechanical the sample Collect the Filtration
cell lysis to tubes Centrifugation supernatant of sample Purification run
Approx.
120–160 min
40 min 40–80 min 40 min
Fig 28. HisTrap FF crude columns save time during purification of histidine-tagged proteins compared with conventional IMAC.
Sample preparation
For direct loading of an unclarified sample, it is critical to obtain good cell lysis in order to avoid
problems with back pressure.
The protocol below has been used successfully in our own laboratories, but other established procedures
may also work.
1. Dilution of cell paste: Add 5 to 10 ml of binding buffer for each gram of cell paste. To prevent the binding
of host cell proteins with exposed histidines, it is essential to include imidazole at a low concentration in
the sample and binding buffer (see Chapter 4).
2. Enzymatic lysis: 0.2 mg/ml lysozyme, 20 µg/ml DNase, 1 mM MgCl2, 1 mM Pefabloc SC or PMSF (final
concentrations). Stir for 30 min at room temperature or 4°C depending on the sensitivity of the target protein.
3. Mechanical lysis*: Sonication on ice, approximately 10 min
or
Homogenization with a French press or other homogenizer
or
Freeze/thaw, repeated at least five times.
* Mechanical lysis time may have to be extended compared with standard protocols to secure an
optimized lysate for sample loading (to prevent clogging of the column and back pressure problems).
Different proteins have different sensitivity to cell lysis, and caution has to be taken to avoid frothing and
overheating of the sample.
4. Adjust the pH of the lysate: Do not use strong bases or acids for pH adjustment (precipitation risk).
Apply the unclarified lysate on the column directly after preparation.
If the sonicated or homogenized unclarified cell lysate is frozen before use, precipitation and
aggregation may increase. New sonication of the lysate can then prevent increased back-
pressure problems when loading on the column.
Buffer preparation
The optimal concentration of imidazole needed in the sample and buffer to obtain the best purity
and yield differs from protein to protein. Under native conditions, 20 to 40 mM imidazole in the
binding buffer is suitable for many proteins; 500 mM imidazole in the elution buffer is most often
sufficient to completely elute the target protein. Use high-purity imidazole as this will give very
low or no absorbance at 280 nm.
As an alternative to elution with imidazole, you can lower the pH to approximately pH 4.5.
(Note that metal ions will be stripped off the medium below pH 4.0.)
Purification
1. Fill the pump tubing or syringe with distilled water. Remove the stopper and connect the column to the
chromatography system tubing, syringe (use the provided Luer connector), or laboratory pump “drop to
drop” to avoid introducing air into the system.
1 ml/min or 5 ml/min for the 1-ml and 5-ml columns, respectively.
5. Apply the unclarified lysate with a pump or syringe. Typical loading volumes of unclarified lysate (highly
dependent on specific sample, sample pretreatment, and temperature at sample loading):
– HisTrap FF crude 1 ml: Up to 100 ml
– HisTrap FF crude 5 ml: Up to 500 ml
Note that the protein binding capacity may also limit the amount of sample that can be applied.
Continuous stirring of the sample during sample loading is recommended to prevent sedimentation.
Sample loading at 4°C may increase the viscosity of the sample. An adverse effect of increased
sample viscosity is that maximum back pressure for the column is reached at a lower sample
volume loading on the column. Large volumes may increase back pressure, making the use of a
syringe more difficult.
6. Wash with binding buffer until the absorbance reaches a steady baseline (generally at least 10 to 15
column volumes).
7. Elute with elution buffer using a step gradient or a linear gradient. For step elution, 5 column volumes of
elution buffer is usually sufficient. A shallow gradient, for example, a linear gradient over 20 column
volumes or more, can separate proteins with similar binding strengths.
Unclarified lysates may cause increased air bubble formation during purification. An attached
flow restrictor in the chromatography system can prevent this if it causes problems. If a flow
restrictor is attached, it is important to change the pressure limit to 0.5 MPa (5 bar) on the
ÄKTAdesign system (where the column and the flow restrictor give a pressure of 0.3 MPa and
0.2 MPa, respectively).

HisTrap FF crude columns with buffers that include reducing agents.
Blank run:
1. Purification of histidine-tagged protein expressed at low levels in Pichia pastoris
using HisTrap FF crude
HisTrap FF crude columns are well-suited for purification of histidine-tagged proteins expressed at low
levels from hosts such as Pichia pastoris. Using HisTrap FF crude columns, highly pure protein can be
obtained directly from unclarified lysates of P. pastoris.
Figure 29 shows the purification of a histidine-tagged Saccharomyces cerevisiae hydrolase expressed
at low levels in P. pastoris. Unclarified sample was loaded directly onto a HisTrap FF crude 5-ml column
without any centrifugation or filtration of the sample. Purity of the protein from the unclarified sample
was high as determined by SDS-PAGE.
A) mAU Column: HisTrap FF crude 5 ml

Sample: Unclarified lysate of YNR064c (Saccharomyces
5000 cerevisiae hydrolase) expressed in P. pastoris
Sample volume: 130 ml
Elution buffer: 20 mM sodium phosphate, 500 mM NaCl,
500 mM imidazole, 3 mM KCl, pH 7.4
3000
Flow rate: 5 ml/min
2000
1000
0
0 10 20 30 40 ml
B)
Mr
97 000
66 000
45 000
30 000
20 100 Lanes
1. LMW markers
14 400
2. Start material
3. Eluted pool (diluted 1:2)
1 2 3 4 4. Eluted pool (diluted 1:4)
Fig 29. (A) Purification of an unclarified sample of histidine-tagged Saccharomyces cerevisiae hydrolase expressed in P. pastoris on
HisTrap FF crude 5 ml. (B) SDS-PAGE under nonreducing conditions (ExcelGel SDS Gradient 8–18) shows the high purity obtained of the
low-level expression protein.
2. Scale-up from 1-ml to 5-ml HisTrap FF crude
An experiment was performed to scale up from 1-ml to 5-ml HisTrap FF crude columns. The sample
was unclarified E. coli extract containing MBP-(His)6, which had been prepared by enzymatic lysis in
combination with homogenization prior to loading on the column. The samples contained approximately
8 and 40 mg of MBP-(His)6 for the 1-ml and 5-ml columns, respectively.
SDS-PAGE shows that the purity and recovery (mg protein/ml medium) of the histidine-tagged protein
purified on the two columns was almost identical.
A) Column: HisTrap FF crude columns, 1 ml and 5 ml

mAU HisTrap FF crude 1 ml Sample: Unclarified E. coli BL21 lysate containing
MBP-(His)6
Flow rate: HisTrap FF crude 1 ml: 1 ml/min
2000 HisTrap FF crude 5 ml: 5 ml/min
1000
0
0 10 20 30 40 50 ml
B)
mAU HisTrap FF crude 5 ml
4000
3000
2000
1000
0
0 50 100 150 200 250 ml
C)
Mr
97 000
66 000
45 000
30 000 Lanes
1. LMW markers
20 100 2. Start material (diluted 1:10)
14 400 3. Flowthrough, 1-ml column (diluted 1:10)
4. Flowthrough, 5-ml column (diluted 1:10)
5. Eluted pool 1-ml column (diluted 1:5)
1 2 3 4 5 6 6. Eluted pool 5-ml column (diluted 1:5)
Fig 30. Scale-up from (A) 1-ml to (B) 5-ml HisTrap FF crude columns. Recovery of protein was 6.3 and 35.2 mg for the 1-ml and 5-ml
columns, respectively. (C) SDS-PAGE under nonreducing conditions (ExcelGel SDS Gradient 8–18) confirms that scaling up from the
1-ml to the 5-ml column does not significantly affect the purification result.
3. Automated, multi-step purification using HisTrap FF crude
HisTrap FF crude columns can be run on ÄKTAdesign systems such as ÄKTAxpress for high-throughput
purification of histidine-tagged proteins. ÄKTAxpress enables automated, parallel purification of histidine-
tagged proteins with the capacity to run a number of different multi-step protocols. A method wizard
supplied with the UNICORN control software makes it easy to create methods for different purification
protocols. Figure 31 shows an automated two-step purification of an unclarified lysate of E. coli
containing MBP-(His)6. The first step in the purification protocol was affinity chromatography (AC), using
HisTrap FF crude 1-ml column. The eluted peak from the affinity step was automatically collected in a
loop and reinjected onto a HiLoad™ 16/60 Superdex 75 pg gel filtration column in the second step of the
purification. Purity of the protein in fractions from the gel filtration step was confirmed by SDS-PAGE
(Fig 31B).
The results show that HisTrap FF crude together with ÄKTAxpress facilitates and enables significant
time savings in the purification of histidine-tagged proteins without compromising sample purity.
A) mAU Affinity chromatography (AC) conditions

A280 Column: HisTrap FF crude 1 ml
2000 Conductivity Sample: Unclarified E. coli BL21 lysate containing
MBP-(His)6
Eluted protein (AC) 500 mM imidazole, pH 7.4
1000 Flow rate: 1 ml/min
System: ÄKTAxpress
Pooled Gel filtration (GF) conditions
5000 fractions (GF)
Column: HiLoad 16/60 Superdex 75 pg
Buffer: 10 mM sodium phosphate, 140 mM NaCl,
0 2.7 mM KCl, pH 7.4
Flow rate: 1.5 ml/min
0 50 100 ml
System: ÄKTAxpress
AC GF
B)
Mr
97 000
66 000
45 000
30 000
Lanes
20 100
1. LMW markers
3. Flowthrough, HisTrap FF crude 1-ml (diluted 1:10)
1 2 3 4 4. Pool from HiLoad 16/60 Superdex 75 pg (diluted 1:2)
Fig 31. (A) Automated two-step purification of MBP-(His)6 from an unclarified lysate of E. coli using HisTrap FF crude and HiLoad 16/60
Superdex 75 pg on ÄKTAxpress. (B) SDS-PAGE was performed under nonreducing conditions on ExcelGel SDS Gradient 8–18.
Purification using a syringe and HisTrap FF crude Kit
HisTrap FF crude Kit is designed for rapid and convenient purification of histidine-tagged proteins using
premade buffers and a syringe. Histidine-tagged proteins can be purified directly from unclarified cell
lysates. This saves time because the pretreatment of the sample is minimized and may increase the
activity of the target protein.
The kit contains three ready-to-use 1-ml HisTrap FF crude columns (containing Ni Sepharose 6 Fast Flow),
buffer concentrates, a 5-ml syringe, and connectors. The kit provides a sufficient volume of buffer
concentrates to perform 10 to 12 purifications when operated with a syringe. The special design of the
column, together with Ni Sepharose 6 Fast Flow, provides fast, easy, and reproducible separations in a
convenient format. Note that HisTrap FF crude columns cannot be opened or refilled.
Direct loading of unclarified cell lysates decreases the total purification time and may increase
the possibility of purifying sensitive target proteins without losing their activity.
Fig 32. HisTrap FF crude Kit provides convenient and simple purification of histidine-tagged proteins.
Sample preparation
may also work.
concentrations). Stir for 30 min at room temperature or 4°C depending on the sensitivity of the target
protein.
or
or
Freeze/thaw, repeated at least five times
Different proteins have different sensitivity to cell lysis, and caution has to be taken to avoid frothing and
overheating of the sample.
4. Adjust the pH of the lysate: Do not use strong bases or acids for pH adjustment (precipitation risk). Apply
the unclarified lysate on the column directly after preparation.
Buffer preparation
Binding buffer: Mix 3 ml of Phosphate buffer 8× stock solution (included in kit) with 0.24 ml of
2 M imidazole (included in kit) and add water to 24 ml. Check pH and adjust to
pH 7.4 to 7.6 if necessary. This buffer now contains 20 mM phosphate,
500 mM NaCl, and 20 mM imidazole.
Elution buffer: Mix 1 ml of Phosphate buffer 8× stock solution (included in kit) with 2 ml of
2 M imidazole (included in kit) and add distilled water to 8 ml. Check pH and adjust
to pH 7.4 to 7.6 if necessary. This buffer now contains 20 mM phosphate,
500 mM NaCl, and 500 mM imidazole.
The high salt concentration in the buffer stock solution may cause salt crystals to form at low
temperature. These crystals will dissolve at room temperature. We therefore recommend that
the buffer stock solutions be allowed to reach room temperature before use. The formation of
salt crystals that dissolve at room temperature does not affect the performance of the product.
Table 10 provides the mixing table for 50 ml of buffer. To obtain the imidazole concentration indicated
in the first column, mix Phosphate buffer 8 × stock solution, 2 M imidazole, and distilled water according
to the table. Check pH and adjust to pH 7.4 to 7.6 if necessary. These buffers will contain 20 mM phosphate,
500 mM NaCl, and the concentrations of imidazole indicated. For one purification, 24 ml of the binding
buffer and 8 ml of each elution buffer are sufficient.
Table 10. Mixing table for 50 ml of buffer.
Imidazole concentration Phosphate buffer 8x 2 M Imidazole Deionized

in buffer stock solution pH 7.4 pH 7.4 water
mM ml ml ml
0 6.25 0 to 50 ml
10 6.25 0.25 to 50 ml
20 6.25 0.50 to 50 ml
30 6.25 0.75 to 50 ml
40 6.25 1.00 to 50 ml
50 6.25 1.25 to 50 ml
60 6.25 1.50 to 50 ml
70 6.25 1.75 to 50 ml
80 6.25 2.00 to 50 ml
90 6.25 2.25 to 50 ml
100 6.25 2.50 to 50 ml
150 6.25 3.75 to 50 ml
200 6.25 5.00 to 50 ml
250 6.25 6.25 to 50 ml
300 6.25 7.50 to 50 ml
400 6.25 10.00 to 50 ml
500 6.25 12.50 to 50 ml

HisTrap FF crude columns with buffers that include reducing agents.
Blank run:
Basic protein purification

When high yield is more important than optimal purity, use the following procedure. When
optimal purity is required, use the optimized procedure (next page) instead.
1. Using the buffer concentrate provided, prepare 24 ml of binding buffer.
2. Using the buffer concentrate provided, prepare 8 ml of elution buffer.
3. Fill the syringe with distilled water. Remove the stopper and connect the column to the syringe with the
provided Luer connector “drop to drop” to avoid introducing air into the column. (If air becomes trapped in
the column, wash it with distilled water until the air disappears.)
4. Remove the snap-off end. Wash the column with 5 ml of distilled water.
5. Using the syringe, equilibrate the column with 5 to 10 ml of binding buffer.
6. Apply the unclarified lysate with the syringe. Collect the flowthrough fraction. A pump (e.g., Peristaltic
Pump P-1) is convenient for large volumes (more than 15 ml) using a maximum flow rate of 3 ml/min.
syringe more difficult. Typical loading volumes of unclarified lysate (highly dependent on specific
sample, sample pretreatment, and temperature at sample loading): Up to 100 ml.
7. Wash with 10 ml of binding buffer. Collect the wash fraction.
8. Elute with 5 ml of elution buffer. Avoid dilution of the eluate by collecting it in 1-ml fractions.
9. Check the different fractions for the purified protein (e.g., by SDS-PAGE and/or Western blotting). The purified
protein is most likely found in the second and third milliliter of the elution step.
For A280 measurement, use the elution buffer as a blank. If imidazole needs to be removed, use
HiTrap Desalting, HiPrep 26/10 Desalting, or PD-10 Desalting Columns.
10. After elution, regenerate the column by washing it with 10 ml of binding buffer. The column is now ready
for a new purification.
For more information on this and on cleaning and storage, refer to Appendix 1.
Optimized protein purification
When optimal purity is needed, the following general procedure for stepwise gradient elution
should be used.
The next time the same protein is to be purified, the number of steps can be reduced to those
described under “Basic protein purification” with the optimal imidazole concentrations selected
here.
1. Prepare binding buffer and five steps of elution buffer ranging from 40 mM to 500 mM imidazole. Check
pH of each after mixing and adjust to pH 7.4 to 7.6 if necessary. See buffer mixing table, page 62.
2. Fill the syringe with distilled water. Remove the stopper and connect the column to the syringe with the
provided Luer connector “drop to drop” to avoid introducing air into the column. (If air becomes trapped in
the column, wash it with distilled water until the air disappears.)
3. Remove the snap-off end. Wash the column with 5 ml of distilled water.
4. Using the syringe, equilibrate the column with 5 to 10 ml of binding buffer.
5. Apply the unclarified lysate with the syringe. Collect the flowthrough fraction. A pump (e.g., Peristaltic
Pump P-1) is convenient for large volumes (more than 15 ml) using a maximum flow rate of 3 ml/min.
syringe more difficult. Typical loading volumes of unclarified lysate (highly dependent on specific
sample, sample pretreatment, and temperature at sample loading): Up to 100 ml.
6. Wash with 10 ml of binding buffer. Collect the wash fraction.
7. Start elution with 5 ml of the first elution buffer containing 40 mM imidazole. Avoid dilution by collecting
the eluate in 1-ml fractions.
8. Proceed with the next imidazole concentration. (For example, elute with 5 ml of elution buffer containing
60 mM imidazole.) Collect the eluate in 1-ml fractions as above.
9. Proceed with the buffers of increasing imidazole concentration, as described in steps 6 and 7. The purified
protein is most likely found in the second and third fraction of one of the elution steps.
10. Check the different fractions for the purified protein (e.g., by SDS-PAGE and/or Western blotting).
For A280 measurements, use the elution buffers as blanks. If imidazole is to be removed, use
HiTrap Desalting, HiPrep 26/10 Desalting, or PD-10 Desalting Columns.
11. After elution, reequilibrate the column with 10 ml of binding buffer. The column is now ready for a new
purification.
The results of the above purification provide information about the optimal binding and elution buffers.
The optimal elution buffer is the one that eluted the histidine-tagged protein. The optimal binding
(wash) buffer is the one from the step before, with a lower concentration of imidazole. Using the highest
possible concentration of imidazole in the binding buffer will give the highest purity of the purified
protein. Use these buffers for the next purification of an identical protein.
The concentration of imidazole needed to prevent nonspecific binding of host cell proteins (without any
elution of histidine-tagged protein) is generally more important to determine than the concentration
needed for elution. A concentration of 500 mM can be used for elution in most cases.
Application example
Purification using HisTrap FF crude Kit
HisTrap FF crude Kit includes three 1-ml HisTrap FF crude columns, ready-made binding and elution
buffer concentrates, connectors, a syringe, and instructions. The kit allows purification in a matter of
minutes starting from an unclarified cell lysate using a simple, four-step procedure:
1. Lyse cells containing histidine-tagged protein.
2. Prepare buffers by mixing and diluting the concentrates.
3. Use the syringe to load unclarified sample to the column, wash, and elute the target protein.
4. Check purity by SDS-PAGE.
As Figure 33 shows, MBP-(His)6 is effectively purified on the HisTrap FF crude 1-ml column using a syringe
and the buffers included in HisTrap FF crude Kit.
A) Imidozole Column: HisTrap FF crude 1 ml
Absorbance (AU) conc. (mM) Sample: MBP-(His)6, Mr 43 000
Elution 3
3.0 500 Flow rate: Approx. 1 ml/min
Buffers: Included in the HisTrap FF crude Kit , prepared
2.5 400 according to the instructions
2.0 Elution 2 Fraction size: 1 ml
300
1.5 Equipment: Manual purification using a syringe
200
1.0
Elution 1
0.5 Wash 3 100
Wash 2
Wash 1
0 0
0 5 10 15 20 25 30
Elution volume (ml)
1 ml/fraction
B)
LMW S W 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30
Mr
97 000
66 000
45 000
30 000
20 100
14 400
Wash 1:10 mM Wash 2:30 mM Wash 3:50 mM Elution 1:100 mM Elution 2:300 mM Elution 3:500 mM
Lanes Lanes
LMW LMW markers Fraction 1–5 Wash 1, 10 mM imidazole in buffer
S Sample, unclarified, diluted 1:10 Fraction 6–10 Wash 2, 30 mM imidazole in buffer
W Wash 5 ml, 10 mM imidazole Fraction 11–15 Wash 3, 50 mM imidazole in buffer
Fraction 16–20 Elution 1, 100 mM imidazole in buffer
Fig 33. (A) Purification of MBP-(His)6 using HisTrap FF crude Kit. (B) Native SDS-PAGE (ExcelGel 8–18) of 1-ml fractions from the purification.
Gravity-flow purification using His GraviTrap and
His GraviTrap Kit
His GraviTrap columns are designed for fast and simple purification of histidine-tagged proteins using
gravity flow. Both clarified and unclarified sample can be applied to the column. The column is
prepacked with Ni Sepharose 6 Fast Flow. Special column frits protect the medium from running dry
during purification. A typical purification run on His GraviTrap is performed in approximately 30 min
(depending on sample volume and viscosity of the solutions).
Fig 34. His GraviTrap connected to LabMate PD-10 Buffer Reservoir for convenient equilibration, sample application, and wash.
His GraviTrap columns are delivered in a package that can be converted into a column stand to simplify
purification. LabMate™ PD-10 Buffer Reservoir can be connected to the columns for convenient
handling of sample volumes above 10 ml. For optimal performance, use His GraviTrap with buffers
prepared from His Buffer Kit.
The benefits of His GraviTrap and His Buffer Kit are combined in His GraviTrap Kit, which contains two
packs of His GraviTrap and one pack of His Buffer Kit. His GraviTrap Kit contains columns and buffers
for 20 purifications.
1. Equilibrate 2. Load sample 3. Wash 4. Elute Total time
0:20 h
Fig 35. Purifying histidine-tagged proteins with His GraviTrap is a simple and quick four-stage procedure.
Sample preparation
may also work.
concentrations). Stir for 30 min at room temperature or 4°C depending on the sensitivity of the target
protein.
or
or
Freeze/thaw, repeated at least five times
Different proteins have different sensitivity to cell lysis, and caution has to be taken to avoid frothing
and overheating of the sample.
4. Adjust the pH of the lysate: Do not use strong bases or acids for pH adjustment (precipitation risk). Apply
the unclarified lysate on the column directly after preparation.
Buffer preparation
Binding buffer: 20 mM sodium phosphate, 500 mM NaCl, 20 mM imidazole, pH 7.4.
the target protein.
Purification
1. Cut off the bottom tip, remove the top cap, pour off excess liquid, and place the column in the Workmate
column stand. If needed, mount LabMate (funnel) on top of the column.
2. Equilibrate the column with 10 ml of binding buffer. The frits protect the column from running dry during
the run.
3. Add 0.5 to 35 ml of the prepared sample.
The protein binding capacity of the column is high (approx. 40 mg histidine-tagged protein/
column); however, the value is protein dependent.
4. Wash with 10 ml of binding buffer.
5. Apply 3 ml of elution buffer and collect the eluate. Under denaturing conditions, elute twice with 3 ml of
elution buffer.
If you use buffers containing denaturing agents or viscous solutions, perform the purification at
room temperature.

His GraviTrap columns with buffers that include reducing agents.
Blank run:
Application example
Rapid purification of a high-molecular-weight histidine-tagged protein using His GraviTrap
His GraviTrap, prepacked with Ni Sepharose 6 Fast Flow, allows quick and simple purification of
histidine-tagged proteins without the need for a pump or purification system. A single column allows
purification of approximately 40 mg of protein in as little as 20 to 25 minutes. Large volumes of clarified
or unclarified samples can easily be applied, and the purified protein can be eluted in a small volume,
resulting in a highly concentrated target protein.
In this example, 20 ml of a clarified E. coli JM109 lysate containing (His)10-TRX-P450 (Mr ~ 130 000) was
purified in just 25 minutes and analyzed by SDS-PAGE and Western blot (Fig 36A–B). SDS-PAGE analysis
shows three major protein bands in the eluted fractions. Western blot analysis and N-terminal
sequencing (data not shown) confirm that each of the three bands in the eluates contains a histidine
tag. The low-molecular-weight bands are truncated forms of the histidine-tagged target protein.
Method:
Equilibration: 10 ml binding buffer (including 40 mM imidazole)
Sample application: 20 ml sample (including 40 mM imidazole)
Wash: 2 × 10 ml binding buffer (including 40 mM imidazole)
Elution: 2 × 3 ml elution buffer
Western blot:
Electrophoresis and transfer: PhastSystem™ and PhastGel™ Gradient 10–15
Membrane: Hybond ECL
Primary antibody: Anti-His antibody (mouse)
Secondary antibody: Anti-mouse IgG, HRP-linked
Detection: Colorimetric, DAB-enhanced liquid substrate
A) B)
Mr
220 000
(His)10-TRX-P450 (His)10-TRX-P450
97 000
66 000 Mr
45 000 45 000
30 000
30 000
20 100
14 400
1 2 3 4 5 6 1 2 3 4 5 6
Lanes
1. High-Range Rainbow™ Molecular Weight Markers
2. Start material (diluted 1:20)
3. Flowthrough (diluted 1:20)
4. Eluate 1
5. Eluate 2
6. Negative control (JM109 nontransformed)
Fig 36. (A) SDS-PAGE and (B) Western blot of purified (His)10-TRX-P450 using His GraviTrap column.
Scale-up purification using HisPrep FF 16/10
HisPrep FF 16/10 columns are specially designed 20-ml HiPrep columns, ready to use for easy, one-
step preparative purification of histidine-tagged proteins. Prepacked with Ni Sepharose 6 Fast Flow,
the columns exhibit high binding capacity and excellent flow properties. For easy scale-up, columns
can be connected in series (back pressure will increase).
Fig 37. HisPrep FF 16/10 column for convenient scale-up purification of histidine-tagged proteins.
The column is made of polypropylene, which is biocompatible and noninteractive with biomolecules.
Purifications can be easily achieved using a chromatography system such as ÄKTAdesign or other
chromatography systems (connectors are included in each package for easy connections). Refer to
Table 8 for a selection guide to purification equipment and to Appendix 1 for a list of HisPrep FF 16/10
column parameters. Note that HisPrep FF 16/10 columns cannot be opened or refilled.
Sample preparation
Chapter 4).
Buffer preparation
the target protein.
Purification
For first-time use, it is important to set an appropriate pressure limit on the system and equilibrate the
column by running 100 ml of binding buffer through it.
1. Apply the centrifuged and/or filtered sample (in binding buffer) to the column at a flow rate of 1 to
10 ml/min (30 to 300 cm/h).
2. Wash the column with 5 to 10 column volumes of binding buffer at 2 to 10 ml/min (60 to 300 cm/h).
3. Elute the bound protein with 5 to 10 column volumes of elution buffer at a flow rate of 2 to 10 ml/min
(60 to 300 cm/h).
4. After elution, regenerate the column by washing it with approximately 100 ml of binding buffer. The

HisPrep columns with buffers that include reducing agents.
Blank run:
Application example
Refer to Application example 3 on page 50.
Purification using uncharged media
Selection Guide — Uncharged IMAC Sepharose products
Will you use

Uncharged an automated What
purification system are your
IMAC Sepharose Yes
requirements
such as
media ÄKTAdesign ?
?
Using
gravity-flow, IMAC Sepharose
No batch 6 Fast Flow
purification
Syringe HiTrap IMAC FF
Contains IMAC Sepharose High Performance.

Contains IMAC Sepharose 6 Fast Flow.
Fig 38. Selection guide for uncharged IMAC Sepharose products.
Figure 38 provides a selection guide for the uncharged IMAC Sepharose products, and Table 11 describes
these options in more detail. In general, IMAC Sepharose High Performance is recommended when high
resolution and high capacity are important, whereas IMAC Sepharose 6 Fast Flow is recommended
when scale-up is required.
Table 11. Purification options for histidine-tagged proteins using uncharged IMAC Sepharose products.

IMAC 25 ml 40 mg/ml For high resolution and + (+) - - +
Sepharose 100 ml (Ni2+) elution of a more
High concentrated sample
Performance (high-performance
purification).
HiTrap 1 ml 40 mg/ For use mainly with a - - - (+) +
IMAC HP column peristaltic pump or
(Ni2+) chromatography system.
5 ml 200 mg/ For high resolution and
column elution of a more
(Ni2+) concentrated sample
(high-performance
purification).
IMAC 25 ml 40 mg/ml Excellent for scale-up due + + + - +
Sepharose 100 ml (Ni2+) to high capacity and high
6 Fast Flow flow properties.
Yes HiTrap IMAC HP
High Prepacked
resolution columns
?
IMAC Sepharose
No
High Performance
Scalability/ Prepacked
high flow columns No IMAC Sepharose 6 Fast Flow
rates
?
>100 mg HiPrep IMAC FF 16/10
Amount
of histidine-
Yes tagged
protein
<100 mg HiTrap IMAC FF
Table 11. Purification options for histidine-tagged proteins using uncharged IMAC Sepharose products (continued).

HiPrep 20 ml 800 mg/ For use with a - - - - +
IMAC FF column chromatography system.
16/10 (Ni2+) Scale-up purification.
HiTrap 1 ml 40 mg/ For use with syringe, - - - + +
IMAC FF column peristaltic pump, or
(Ni2+) chromatography system.
5 ml 200 mg/ Provides excellent flow
column properties.
(Ni2+) Scale-up.
Contains IMAC Sepharose High Performance
Contains IMAC Sepharose 6 Fast Flow
Purification using IMAC Sepharose High Performance
IMAC Sepharose High Performance is an uncharged medium consisting of 34-µm beads of highly
cross-linked 6% agarose to which a chelating group has been covalently coupled. This chelating group
will be charged with suitable metal ions by the user, allowing the medium to selectively retain target
proteins. The small bead size allows high chromatographic resolution with distinctly separated peaks
containing concentrated material. The medium is highly compatible with a range of additives and is
well suited to high-performance purifications that produce concentrated products in the eluate. Refer
to Appendix 1 for a list of the characteristics of IMAC Sepharose High Performance. IMAC Sepharose
High Performance is supplied preswollen in 20% ethanol.
Fig 39. IMAC Sepharose High Performance is supplied free of metal ions, enabling it to be used across a range of applications for purifying
histidine-tagged as well as native proteins. It is available in 25 ml and 100 ml lab packs as well as prepacked HiTrap IMAC HP 1-ml and
5-ml columns.
Packing a column
Ideally, IMAC Sepharose High Performance media are packed in XK or Tricorn columns in a
two-step procedure: Do not exceed 1.0 bar (0.1 MPa) in the first step and 3.5 bar (0.35 MPa) in
the second step. If the packing equipment does not include a pressure gauge, use a packing
flow rate of 5 ml/min (XK 16/20 column) or 2 ml/min (Tricorn 10/100 column) in the first step,
and 9 ml/min (XK 16/20 column) or 3.6 ml/min (Tricorn 10/100 column) in the second step. If the
recommended pressure or flow rate cannot be obtained, use the maximum flow rate your pump
can deliver in order to achieve a well-packed bed.
water.
Sample preparation
Chapter 4).
Buffer preparation
Binding buffer: 20 mM sodium phosphate, 0.5 to 1.0 M NaCl, 20 to 40 mM imidazole, pH 7.4. (The optimal
Elution buffer: 20 mM sodium phosphate, 0.5 M NaCl, 500 mM imidazole, pH 7.4.
a 0.22 µm or a 0.45 µm filter before use. High-purity imidazole will give low to no absorbance at
280 nm.
Charging the medium with metal ion

1. Prepare a 0.1 M solution of the desired metal ion (e.g., Cu2+, Zn2+, Co2+, Fe3+, or Ni2+) in distilled water.
Salts of chlorides, sulfates, etc., can be used (e.g., 0.1 M CuSO4 or 0.1 M NiSO4).
Solutions of Zn2+ ions should have a pH of approximately 5.5 or lower to avoid solubility problems
that arise at pH 6 or higher. Fe3+ ions should be immobilized at low pH (approximately pH 3.0) to
avoid formation of insoluble ferric compounds.
2. Wash the column with at least 2 column volumes of distilled water.
3. Apply at least 0.2 column volumes of the metal ion solution to the column.
4. Wash the column with at least 5 column volumes of distilled water to remove excess metal ions.
Washing with buffer before applying the metal ion solution may cause unwanted precipitation.
IMAC Sepharose High Performance is compatible with reducing agents. However, we recommend
removal of any weakly bound metal ions before applying buffer/sample that includes reducing
agents. This can be accomplished by performing a blank run without reducing agents (see below).
Do not store IMAC Sepharose High Performance with buffers that include reducing agents.
Leakage of metal ions is low under all normal conditions. For critical applications, leakage
during purification can be diminished by performing a blank run (as described below) before
loading sample.
Blank run:
Purification
1. If necessary, wash out the 20% ethanol with 5 column volumes of distilled water. Use a linear flow rate of
50 to 100 cm/h. Refer to Appendix 6 for flow rate calculations.
4. Wash the column with binding buffer until the absorbance reaches the baseline.
column.
Purification using IMAC Sepharose 6 Fast Flow
IMAC Sepharose 6 Fast Flow consists of 90-µm beads of highly cross-linked agarose to which a chelating
group has been covalently coupled. This chelating group will be charged with suitable metal ions by
the user, allowing the medium to selectively retain target proteins.
IMAC Sepharose 6 Fast Flow displays a high protein binding capacity. The binding capacity is protein
dependent and metal-ion dependent. The medium is easy to pack and use, and its high flow properties
make it excellent for scaling up. Refer to Appendix 1 for a list of the characteristics of this medium.
IMAC Sepharose 6 Fast Flow is supplied preswollen in 20% ethanol.
Fig 40. IMAC Sepharose 6 Fast Flow provides high flow properties to enable scaled-up purification of histidine-tagged and native
proteins. It provides numerous possibilities for optimizing purifications at both laboratory and process scale.
Packing a column
Ideally, IMAC Sepharose 6 Fast Flow media are packed in XK or Tricorn columns in a two-step
procedure: Do not exceed 0.5 bar (0.05 MPa) in the first step and 1.5 bar (0.15 MPa) in the
second step. If the packing equipment does not include a pressure gauge, use a packing flow
rate of 2.5 ml/min (XK 16/20 column) or 0.9 ml/min (Tricorn 10/100 column) in the first step, and
8.7 ml/min (XK 16/20 column) or 4.7 ml/min (Tricorn 10/100 column) in the second step.
water.
Sample preparation
Chapter 4).
Buffer preparation
Binding buffer: 20 mM sodium phosphate, 0.5 to 1.0 M NaCl, 20 to 40 mM imidazole, pH 7.4. (The optimal
Elution buffer: 20 mM sodium phosphate, 0.5 M NaCl, 500 mM imidazole, pH 7.4.
280 nm.
sufficient to completely elute the target protein.
Charging the medium with metal ion

1. Prepare a 0.1 M solution of the desired metal ion (e.g., Cu2+, Zn2+, Co2+, Fe3+, or Ni2+) in distilled water.
Salts of chlorides, sulfates, etc., can be used (e.g., 0.1 M CuSO4 or 0.1 M NiSO4).
IMAC Sepharose 6 Fast Flow is compatible with reducing agents. However, we recommend
removal of any weakly bound metal ions before applying buffer/sample that includes reducing
agents. This can be accomplished by performing a blank run without reducing agents (see
below). Do not store IMAC Sepharose 6 Fast Flow with buffers that include reducing agents.
Leakage of metal ions is low under all normal conditions. For critical applications, leakage
during purification can be diminished by performing a blank run (as described below) before
loading sample.
Blank run:
Purification
1. If necessary, wash out the 20% ethanol with 5 column volumes of distilled water. Use a linear flow rate of
50 to 100 cm/h. Refer to Appendix 6 for flow rate calculations.
4. Wash the column with binding buffer until the absorbance reaches the baseline.
column.
Purification using HiTrap IMAC HP and
HiTrap IMAC FF columns
HiTrap IMAC HP and HiTrap IMAC FF are 1-ml and 5-ml columns prepacked with IMAC Sepharose High
Performance or IMAC Sepharose 6 Fast Flow, respectively. Sample application, washing, and elution can
be performed using a syringe with a supplied adapter, a peristaltic pump, or a liquid chromatography
system such as ÄKTAdesign (see Table 8 for equipment choices).
HiTrap IMAC HP and HiTrap IMAC FF columns are made of polypropylene, which is biocompatible and
noninteractive with biomolecules. The top and bottom frits are manufactured from porous polyethylene.
Columns are delivered with a stopper on the inlet and a snap-off end on the outlet. Each package
includes all necessary components for connecting the columns to different types of equipment. For
quick scale-up of purifications, two or three HiTrap columns (1 ml or 5 ml) can be connected in series
(back pressure will be higher). Note that HiTrap IMAC columns cannot be opened or refilled.
Fig 41. HiTrap IMAC HP 1-ml columns charged with Cu2+, Zn2+, Co2+ and Ni2+, respectively.
Sample preparation
Chapter 4).
Buffer preparation
280 nm.
Charging the column with metal ion
1. Prepare a 0.1 M solution of the desired metal ion (e. g., Cu2+, Zn2+, Co2+, Fe3+, or Ni2+) in distilled water. Salts
of chlorides, sulfates, etc., can be used (e.g., 0.1 M CuSO4 or 0.1 M NiSO4).
2. Fill the pump tubing or syringe with distilled water. Remove the stopper and connect the column to the
chromatography system tubing, syringe (use the connector supplied), or laboratory pump “drop to drop”
to avoid introducing air into the system.
4. Wash out the ethanol with 5 ml (HiTrap IMAC HP or FF 1-ml columns) or 15 ml (HiTrap IMAC HP or FF 5-ml
columns) of distilled water. Do not use buffer to wash the column at this stage; a buffer wash may cause
the metal ion to precipitate during step 5.
5. Charge the water-washed column by loading at least 0.5 ml (HiTrap IMAC HP or FF 1-ml columns) or
2.5 ml (HiTrap IMAC HP or FF 5-ml columns) of 0.1 M metal ion/salt solution.
6. Repeat the water wash described in step 4.
1 ml/min or 5 ml/min for the 1-ml and 5-ml columns, respectively.
IMAC Sepharose is compatible with reducing agents. However, we recommend removal of any
weakly bound metal ions before applying buffer/sample that includes reducing agents. This can
HiTrap IMAC columns with buffers that include reducing agents.
For critical applications, leakage of metal ions during purification can be diminished by performing
a blank run (as described below) before loading sample.
Blank run:
Purification
1. Apply the pretreated sample using a syringe fitted to the connector or by pumping it onto the column.
sample application*.
steady baseline.
3. Elute with elution buffer using a one-step or linear gradient. For step elution, 5 column volumes usually
suffice. A linear gradient over 20 column volumes or more may separate proteins with similar binding
strengths.
Application example
Screening for optimized purity using different metal ions
YNR064c (Mr 33 700) is a (histidine)6-tagged protein expressed in Pichia pastoris. It was purified using
HiTrap IMAC HP 1-ml columns charged separately with Cu2+, Zn2+, Co2+, or Ni2+; conditions were
otherwise the same for the four purifications. See Figures 42A-E for the resulting chromatograms and
SDS-PAGE analysis of pooled fractions.
The results show that for this (histidine)6-tagged target protein, the highest purity was achieved with
Ni2+ or Cu2+, although Cu2+, at the conditions used, apparently gave a small loss of target protein
(Figure 42E, lane 4).
%B Column: HiTrap IMAC HP 1 ml, charged with:

mAU A) Cu2+ B) Zn2+ C) Co2+ D) Ni2+
A) Cu2+ Conditions were otherwise the same for
4000 80 the four purifications
Sample: Histidine-tagged YNR064c (Mr 33 700) in
3000 60 clarified Pichia pastoris extract including
20 mM imidazole
2000 40 Sample volume: 10 ml
0.0 10.0 20.0 30.0 40.0 50.0 60.0 ml
Cu2+
Flow rate: 1 ml/min

%B
mAU pool Gradient: 4–60% elution buffer (20–300 mM imidazole)
in 25 ml 100% elution buffer
B) Zn2+
4000 80 (500 mM imidazole) in 5 ml
Detection: Absorbance, 280 nm
3000 60 System: ÄKTAexplorer 10
2000 40
1000 20
0 0
0.0 10.0 20.0 30.0 40.0 50.0 60.0 ml E)
Zn 2+
pool %B
mAU Mr
C) Co2+ 97 000
4000 80 66 000
3000 60 45 000
30 000
2000 40
20 100
20 14 400
1000
0 0
0.0 10.0 20.0 30.0 40.0 50.0 60.0 ml
Co2+
Lanes 1 2 3 4 5 6 7 8 9 10 11 12
%B
mAU pool 1. LMW markers
D) Ni 2+ 2. Start material, diluted 1:10
4000 80 3. Flowthrough, diluted 1:10, Cu2+
3000 4. Wash, Cu2+
60
5. Eluted pool, Cu2+
2000 40 6. Wash, Zn2+
7. Eluted pool, Zn2+
1000 20 8. Wash, Co2+
9. Eluted pool, Co2+
0 0 10. Wash, Ni2+
0.0 10.0 20.0 30.0 40.0 50.0 60.0 ml
11. Eluted pool, Ni2+
Ni 2+
pool 12. LMW markers

Fig 42. Purification of (histidine)6-tagged YNR064c expressed in Pichia pastoris on four different HiTrap IMAC HP 1-ml columns charged
separately with metal ions (A) Cu2+, (B) Zn2+, (C) Co2+, or (D) Ni2+. Pools selected after SDS-PAGE of individual 1-ml fractions (not shown) are
indicated. (E) SDS-PAGE analysis: reducing conditions on ExcelGel SDS Gradient 8–18; Coomassie staining.
Preparative purification using HiPrep IMAC FF 16/10 column
HiPrep IMAC FF 16/10 is a ready-to-use 20-ml column, prepacked with uncharged IMAC Sepharose 6
Fast Flow. The column is well-suited for preparative purification of histidine-tagged recombinant proteins
and untagged, naturally occurring proteins. HiPrep IMAC FF 16/10 provides fast, simple, and easy
separations in a convenient format, and the IMAC Sepharose 6 Fast Flow medium is well-suited for
scaling up.
Separations can be easily achieved using a chromatography system such as ÄKTAdesign. Refer to
Table 8 for a selection guide to purification equipment and to Appendix 1 for a list of HiPrep IMAC FF
16/10 column parameters.
IMAC Sepharose 6 Fast Flow is also available as prepacked 1-ml and 5-ml HiTrap IMAC FF columns and
as a bulk medium in lab packs (25 and 100 ml) for packing columns.
Sample preparation
buffer; or by buffer exchange. To prevent the binding of host cell proteins with exposed
histidines, it is essential to include imidazole at a low concentration in the sample and binding
buffer (see Chapter 4).
Buffer preparation
280 nm.
Charging the column with metal ion

1. Prepare a 0.1 M solution of the desired metal ion (e. g., Cu2+, Zn2+, Co2+, Fe3+, or Ni2+) in distilled water. Salts
of chlorides, sulfates, etc., can be used (e.g., 0.1 M CuSO 4 or 0.1 M NiSO4).
Purification
1. Apply the centrifuged and/or filtered sample (in binding buffer) to the column at a flow rate of 1 to
10 ml/min (30 to 300 cm/h).
2. Wash the column with 5 to 10 column volumes of binding buffer at 2 to 10 ml/min (60 to 300 cm/h).
3. Elute the bound protein with 5 to 10 column volumes of elution buffer at a flow rate of 2 to10 ml/min
(60 to 300 cm/h).
4. After elution, regenerate the column by washing it with 5 to 10 column volumes of binding buffer.
The column is now ready for a new purification.
IMAC Sepharose is compatible with reducing agents. However, we recommend removal of any
weakly bound metal ions before applying buffer/sample that includes reducing agents. This can
HiPrep IMAC FF 16/10 columns with buffers that include reducing agents.
For critical applications, leakage of metal ions during purification can be diminished by performing
a blank run (as described below) before loading sample.
Blank run:
Application example
Optimization and scale-up of untagged protein
The IMAC technique can also be used to purify proteins that are not histidine-tagged. This application
presents one such example. Experiments were performed to optimize the method and assess the efficiency
of scaling up the capture step in the purification of recombinant bovine carbonic anhydrase II (r-BCA,
Mr 30 000), a protein that naturally contains exposed histidine residues.
Three metal ions (Cu2+, Ni2+, and Zn2+) and two elution methods (imidazole and pH) were tested to
establish optimal conditions for purifying r-BCA in process-scale applications. HiTrap IMAC FF 1-ml
columns were used to establish the conditions. High purity was obtained with all three metal ions tested;
binding strength decreased in the order Zn2+ = Ni2+ > Cu2+ (data not shown). Zn2+ is the preferred metal
ion for process-scale purification because of its low toxicity, making it the appropriate choice for the
scale-up experiments. Results also showed excellent recovery and purity in both elution methods (data
not shown). Because pH elution is less expensive, it was chosen for the scale-up experiments.
In the scale-up studies, yields were very good (> 90%) with both HiTrap IMAC FF 5-ml and HiPrep IMAC FF
16/10, 20-ml columns (Fig 43). The loading was 74% of maximum binding capacity. No significant
change in recovery and purity was seen between the different scales (Table 12). The recovery of the
enzymatic activity was determined using an esterase activity assay and was found to be approximately
90% in all cases.
Metal ion leakage from the medium, an important concern in industrial applications, was also investigated
in this study. Total leakage of Zn2+ was found to be very low, less than 3% in the HiPrep IMAC FF 16/10
scale. It should be noted that r-BCA needs one zinc ion in the active site for its enzymatic activity. A simple
desalting step (HiPrep 26/10 Desalting) after purification removes all metal ions (except the one
anchored to the active site of the protein).
mAU A) pH Columns: HiTrap IMAC FF (1 ml and 5 ml) and HiPrep
2+
7.5 IMAC FF 16/10 (20 ml) charged with Zn
5000
Sample: 2.4, 12 and 49 ml of clarified E. coli extract
7.0 containing 12.5, 62.4 and 255 mg r-BCA,
4000 respectively
HiTrap IMAC FF 1 ml 6.5 Binding buffer: 20 mM sodium phosphate, 0.5 M NaCl, pH 7.4
Elution buffer: 20 mM sodium acetate, 0.5 M NaCl, pH 4.0
3000 6.0
Flow rate: 150 cm/h in all cases
5.5 Experimental: After sample application, each column was
2000 washed with 20 column volumes (CV) binding
buffer followed by stepwise elution with 15 CV
5.0
100% elution buffer.
1000 Detection: Absorbance, 280 nm
4.5
0
4.0
0.0 5.0 10.0 15.0 20.0 25.0 30.0 ml
Pool
eluate
mAU B) pH mAU C) pH
7.5 5000 7.5
5000
7.0 7.0
4000 4000
HiTrap IMAC FF 5 ml 6.5 HiPrep IMAC FF 16/10, 20 ml 6.5
3000 6.0 3000 6.0
5.5 5.5
2000 2000
5.0 5.0
1000 1000
4.5 4.5
0 0 4.0
4.0
0 50 100 150 ml 0 100 200 300 400 500 600 ml
eluate
Pool
Pool
eluate
D)
Mr
97 000 Lanes
1. LMW markers
66 000
2. Start material, clarified E. coli extract, diluted 1:33
45 000 3. Flowthrough HiTrap IMAC FF 1 ml, diluted 1:4
30 000 4. EIuted pool HiTrap IMAC FF 1 ml, diluted 1:4
5. Flowthrough HiTrap IMAC FF 5 ml, diluted 1:4
20 100 6. EIuted pool HiTrap IMAC FF 5 ml, diluted 1:5
14 400 7. Flowthrough HiPrep IMAC FF 16/10, 20 ml, diluted 1:4
1 2 3 4 5 6 7 8 8. EIuted pool HiPrep IMAC FF 16/10, 20 ml, diluted 1:4
Fig 43. Chromatograms showing scale-up of purification from (A) HiTrap IMAC FF 1-ml column to (B) HiTrap IMAC FF 5-ml column and
(C) HiPrep IMAC FF 16/10 20-ml column. Sample was 2.4, 12, and 49 ml of clarified extract of E. coli containing 12.5, 62.4, and 255 mg of
r-BCA, respectively. The load was approximately 74% of maximum binding capacity. (D) Nonreduced SDS-PAGE analysis on ExcelGel
Gradient 8–18 of the main fractions from the scale-up experiments. The gel was stained with a 1% solution of PhastGel Blue R
(Coomassie).
Table 12. Data and results from the scale-up purification of r-BCA on IMAC Sepharose 6 Fast Flow. Comparisons of r-BCA yields and
recoveries for the different runs show scalability of the application.
Column Fraction Amount Amount Recovery Recovery

applied eluted of of r-BCA
(mg) (mg) protein activity
HiTrap IMAC FF 1 ml Clarified E. coli extract 12.5 - - -
Eluted pool - 11.7 94% 93%
HiTrap IMAC FF 5 ml Clarified E. coli extract 62.4 - - -
Eluted pool - 56.1 90% 84%
HiPrep IMAC FF Clarified E. coli extract 255 - - -
16/10 (20 ml) Eluted pool - 235 92% 90%
Detection of histidine-tagged proteins
Table 13 reviews the methods available for detection of histidine-tagged proteins. These methods can
be selected according to the experimental situation. For example, SDS-PAGE analysis, performed
frequently during expression and purification to monitor results, may not be the method of choice for
routine monitoring of samples from high-throughput screening. Functional assays specific for the
protein of interest are useful but not often available.
Table 13. Detection methods for histidine-tagged proteins.
Generic detection method Comments

(detects the tag)
ELISA assay using anti-His antibody Highly specific, detects only histidine-tagged protein.
Western blot analysis using Highly specific, detects only histidine-tagged protein.
anti-His antibody and ECL Little or no background when used at optimized concentrations
detection systems with secondary HRP-conjugated antibody.
ECL detection systems enhance detection in Western blot.
ECL provides adequate sensitivity for most recombinant expression
applications.
For higher sensitivity use ECL Advance™.
Detection methods specific for
the target protein
SDS-PAGE with Coomassie Provides information on size and % purity.
or silver staining Detects tagged protein and contaminants.
Functional assays Useful to assess if the purified histidine-tagged protein is active.
Not always available.
May require development and optimization.
SDS-PAGE analysis
6X SDS loading buffer: 0.35 M Tris-HCl, 10.28% (w/v) SDS, 36% (v/v) glycerol, 0.6 M dithiothreitol
(or 5% β-mercaptoethanol), 0.012% (w/v) bromophenol blue, pH 6.8.
Store in 0.5 ml aliquots at -80°C.
1. Add 2 µl of 6X SDS loading buffer to 5 to 10 µl of supernatant from crude extracts, cell lysates or purified
fractions as appropriate.
2. Vortex briefly and heat for 5 min at 90°C to 100°C.
3. Load the samples onto an SDS-polyacrylamide gel.
4. Run the gel for the appropriate length of time and stain with Coomassie Blue (Coomassie Blue R Tablets)
or silver (PlusOne Silver Staining Kit, Protein).
The percentage of acrylamide in the SDS-gel should be selected according to the expected
molecular weight of the protein of interest (see Table 14).
Table 14. Separation size range for different percentages of acrylamide in the SDS-PAGE gel.
% Acrylamide in resolving gel Separation size range (Mr x 103)

Single percentage: 5% 36–200
7.5% 24–200
10% 14–200
12.5% 14–100
15% 14–601
Gradient: 5–15% 14–200
5–20% 10–200
10–20% 10–150
1 The larger proteins fail to move significantly into the gel.
Western blot analysis
Expression and purification can be monitored by Western blot analysis using ECL, ECL Plus™, or ECL
Advance detection systems to enhance sensitivity, if required.
Anti-His Antibody
Blocking/Incubation buffer: 5% (w/v) nonfat dry milk and 0.1% (v/v) Tween 20 in PBS (140 mM NaCl,
2.7 mM KCl, 10 mM Na2HPO4, 1.8 mM KH2PO4, pH 7.3)
Wash buffer: 0.1% v/v Tween 20 in PBS (as above)
Secondary Antibody to detect the anti-His antibody (such as antibody to mouse Ig, HRP-linked Whole Ab,
NA931).
1. Separate the protein samples by SDS-PAGE.
Anti-His antibody from GE Healthcare is a monoclonal preparation avoiding the presence of low
levels of cross-reacting antibodies. However, it is recommended to always run a sample of an
E. coli sonicate that does not contain a recombinant histidine-tagged plasmid as a control.
2. Transfer the separated proteins from the electrophoresis gel to an appropriate membrane, such as
Hybond ECL (for subsequent ECL detection) or Hybond P (for subsequent ECL, ECL Plus, or ECL Advance
detection).
Electrophoresis and protein transfer may be accomplished using a variety of equipment and
reagents. For further details, refer to the Protein Electrophoresis Technical Manual and the
Hybond ECL instruction manual from GE Healthcare.
Blocking of membrane
1. Transfer the membrane onto which the proteins have been blotted to a container such as a Petri dish.
2. Add 50 to 200 ml of blocking/incubation buffer.
3. Incubate for 1 to 16 h at ambient temperature with gentle shaking.
4. Decant and discard the buffer.
Longer incubation times (up to 16 h) with blocking buffer may reduce background signal.
Incubation of membrane blot with primary antibody

1. Prepare an appropriate dilution of anti-His antibody with blocking/incubation buffer, for example,
5 to 10 µl of antibody to 50 ml of buffer. Refer to GE Healthcare Application Note 18-1139-13 for further
information on optimization.
2. Pour the antibody-buffer mixture into the container with the membrane.
3. Incubate for 1 h at ambient temperature with gentle shaking.
4. Decant and discard the antibody-buffer.
5. Rinse twice with 20 to 30 ml of blocking or wash buffer to remove most of the unbound antibody.
6. Decant and discard the rinses.
7. Wash the membrane with 20 to 30 ml of blocking or wash buffer for 10 to 60 min at ambient temperature
with gentle shaking.
8. Discard the wash and repeat.
Incubation of membrane blot with secondary antibody
1. Dilute an appropriate anti-mouse secondary antibody with blocking/incubation buffer according to the
manufacturer's recommendation. Refer to GE Healthcare Application Note 18-1139-13 for further
information on optimization.
5. Rinse twice with 20 to 30 ml of blocking or wash buffer to remove most of the unbound antibody.
6. Decant and discard the rinses.
7. Wash the membrane with 20 to 30 ml of blocking or wash buffer for 10 to 60 min at ambient temperature
with gentle shaking.
8. Discard the wash and repeat.
9. Develop the blot with the appropriate substrate for the conjugated secondary antibody.
Refer to GE Healthcare Application Note 18-1139-13 and product brochure 14-0003-87 for
further information on optimization of antibody concentration for Western blotting.
ECL, ECL Plus, and ECL Advance detection systems require very little antibody to achieve a
sufficient sensitivity, so the amount of antibody (primary and secondary) used in the protocols
can be minimized. Smaller quantities of antibody-buffer mixtures can be used by scaling down
the protocol and performing the incubations in sealable plastic bags.
Anti-His antibody from GE Healthcare is a monoclonal preparation and has been tested for its
lack of nonspecific background binding in a Western blot. Some sources of the anti-His antibody
may contain antibodies that react with various E. coli proteins present in the tagged protein
sample. Such antibodies can be removed by cross-absorbing the antibody with an E. coli
sonicate to remove anti-E. coli antibodies. This E. coli should not contain a histidine-tag-
encoding plasmid.
Tag removal by enzymatic cleavage
In most cases, functional tests can be performed using the intact histidine-tagged protein. If removal
of the tag is necessary, then procedures similar to GST tag removal can be followed, that is, specific
recognition sites are incorporated to allow subsequent enzymatic cleavage. The precise protocols
required for cleavage and purification will depend on the original vectors and the properties of the
specific enzymes used for cleavage.
rTEV protease (Invitrogen) has a (histidine)6-tag and recognizes the amino acid sequence
Glu-Asn-Leu-Tyr-Phe-Gln↓Gly. Glu, Tyr, Gln and Gly are needed for cleavage between the Gln
and Gly residues (↓). N-terminal (histidine)6-tags can be removed. The advantage of this enzymatic
cleavage is that the protein of interest can be repurified using the same Ni Sepharose medium
or prepacked column. The (histidine)6-tag and the (histidine)6-tag rTEV protease will both bind to
the column, and the protein of interest can be collected in the flowthrough.
The amount of enzyme, temperature, and length of incubation required for complete digestion
vary according to the specific tagged protein produced. Determine optimal conditions in
preliminary experiments.
Remove samples at various time points and analyze by SDS-PAGE to estimate the yield, purity,
and extent of digestion. Approximate molecular weights for SDS-PAGE analysis:
rTEV protease Mr 29 000
Carboxypeptidase A* Mr 94 000
* for the removal of C-terminal (histidine)6-tags.
There is no PreScission Protease recognition site available for use with histidine-tagged proteins.
Some cleavage procedures will require a second purification step to be performed to remove
the protease or other contaminants. Conventional chromatographic separation techniques
such as gel filtration (usually no need for optimization), ion exchange, or hydrophobic interaction
chromatography will need to be developed (see Appendix 9).
Application example
Automatic histidine tag removal using ÄKTAxpress
Below we present an example of automated tag removal using ÄKTAxpress. All multistep purification
protocols in ÄKTAxpress can be combined with automated on-column tag cleavage. Tag cleavage is
always performed on the affinity column prior to further purification steps. When the cleaved protein
has been eluted, the affinity column is regenerated and affinity tag, tagged protease, and remaining
uncleaved protein are collected in a separate outlet. The procedure involves binding the tagged protein,
injection of protease, incubation, elution of cleaved protein, and collection in capillary loop(s), followed
by further purification steps.
Four-step protocol: (histidine)6-tagged protein cleaved with AcTEV protease

The example in Figure 44 shows purification results for a (histidine)6-tagged protein, APC234 (Mr 32 500),
expressed in E. coli. The Mr of the cleaved product is 30 000. After harvest, cell lysis was performed by
sonication. The samples were clarified by centrifugation prior to sample loading.
Affinity chromatography (AC), desalting (DS), ion exchange (IEX), and gel filtration (GF) were all performed
on ÄKTAxpress using columns as indicated in the figure. The purity of each sample was analyzed by
SDS-PAGE (Coomassie staining). The reduced samples were applied on an SDS-polyacrylamide gel.
Approximately 7.5 µg of protein was loaded per lane.
Columns: AC: HisTrap HP, 5 ml
DS: HiPrep 26/10 Desalting
IEX: RESOURCE™ Q, 6 ml
GF: HiLoad 16/60 Superdex 75 pg
Sample: APC234, Mr 32 000 (cleaved product , Mr 30 000)
Cleavage conditions: 200 units of AcTEV™ protease/mg protein, 8 h incubation time at room temperature
AC binding buffer: 50 mM Tris-HCl, 500 mM NaCl, 20 mM imidazole, pH 7.5
AC cleavage buffer: 50 mM Tris-HCl, 500 mM NaCl, 50 mM imidazole, pH 7.5
AC elution buffer: 50 mM Tris-HCl, 500 mM NaCl, 500 mM imidazole, pH 7.5
DS and IEX binding buffer: 50 mM Tris-HCl, pH 8.0
IEX elution buffer: 50 mM Tris-HCl, 1 M NaCl, pH 8.0
GF buffer: 50 mM Tris-HCl, 150 mM NaCl, pH 7.5
A) B) 1 2 3 4 5
A 280 Mr
mAU
Cleaved protein 97 000
66 000
2000 Regeneration
45 000
30 000
1500
20 100
1000 16 mg
14 400
500 Lanes
1. LMW marker
2. Start sample
3. Flowthrough
0 4. Purified cleaved APC234
0 100 200 300 400 ml
AC DS IEX GF 5. Reference: uncleaved APC234
Fig 44. (A) Four-step protocol for purification of (histidine)6-tagged protein cleaved with AcTEV protease. (B) Analysis of the cleaved
purified protein using SDS-PAGE. The gel was stained with Coomassie.
Troubleshooting
The troubleshooting guide below addresses problems common to the majority of purification products
discussed in this chapter, as well as problems specific to a particular method. In the latter case, the
relevant product is indicated.
Problem Possible cause Solution

Column or plate wells Cell debris is present. Centrifuge and/or pass the sample through a
have clogged 0.22 or 0.45 µm filter.
OR Clean the media according to Appendix 1.
Liquid not completely If cleaning-in-place is unsuccessful, replace the
removed during media/prepacked column. Optimize sample
centrifugation pretreatment before the next sample loading.
(His SpinTrap) Try using HisTrap FF crude columns.
OR
Flow rate is too slow
(His GraviTrap)
The sample is too viscous due Increase dilution of the cell paste before lysis, or
to too high a concentration of dilute after lysis.
material or the presence of large Increase time for lysis until the viscosity is
nucleic acid molecules (may be reduced, and/or add an additional dose of
evidenced by increased back DNase and Mg2+ (DNase I to 5 µg/ml, Mg2+ to
pressure). 1 mM), and incubate on ice for 10 to 15 min.
Increase the efficiency of the mechanical cell
disruption (e.g., increase sonication time).
Keep the sample on ice to avoid frothing and
overheating as this may denature the target
protein. Over-sonication can also lead to
copurification of host proteins with the target
protein.
Freeze/thaw of the unclarified lysate may
increase precipitation and aggregation.
Sonication of the thawed lysate can prevent
increased back pressure problems when loading
on the column.
If the purification has been performed at 4°C,
move to room temperature if possible.
Draw the lysate through a syringe needle
several times.
Decrease the flow rate during sample loading.
Protein is difficult to dissolve or First, screen for suitable conditions for solubility;
precipitates during purification. vary pH, ionic strength, protein concentration,
detergent, other additives that may affect
solubility of the protein. If the protein cannot be
kept in solution by these means, consider using
more harsh conditions, such as 8 M urea,
6 M Gua-HCl, or SDS (or other harsh detergent).
Add detergents, reducing agents or other
additives to the sample [2% Triton X-100,
2% Tween 20, 2% Nonidet™ P-40, 2% cholate,
1% CHAPS, 1.5 M NaCl, 50% glycerol,
20 mM β-mercaptoethanol, 1 to 3 mM DTT or
DTE (up to 5 mM is possible but depends on the
sample and the sample volume), 5 mM TCEP,
10 mM reduced glutathione, 8 M urea, or
6 M Gua-HCl] and mix gently for 30 min to
solubilize the tagged protein.
Protein is difficult to dissolve or Note that Triton X-100 and NP-40 (but not
precipitates during purification. Tween) have a high absorbance at 280 nm.
Furthermore, detergents cannot be easily
removed by buffer exchange.
Inclusion bodies: the protein can usually be
solubilized (and unfolded; refolding needed to
obtain active protein) from inclusion bodies using
common denaturants such as 4 to 6 M Gua-HCl,
4 to 8 M urea, or strong detergents. Mix gently
for 30 min or more to aid solubilization of the
tagged protein. Purify in the presence of the
denaturant.
If possible, decrease the NaCl concentration in the
elution buffer adjust ion strength or pH of sample.
No or low yield of Elution conditions are too mild Elute with increasing imidazole concentration or
histidine-tagged (histidine-tagged protein still decreasing pH to determine the optimal elution
protein in the bound). conditions.
purified fractions
Protein has precipitated in the For the next experiment, decrease amount of
column or wells. sample, or decrease protein concentration by
eluting with linear imidazole gradient instead of
imidazole steps.
Try detergents or changed NaCl concentration,
or elute under denaturing (unfolding) conditions
(use 4 to 8 M urea or 4 to 6 M Gua-HCl).
Nonspecific hydrophobic or other Add a nonionic detergent to the elution buffer
interactions are occurring. (e.g., 0.2% Triton X-100) or increase the NaCl
concentration.
The histidine-tagged The concentration of imidazole in Alter the imidazole concentration—it may be
protein is found in the the sample and/or binding buffer too high.
flowthrough is incorrect.
The histidine tag may be Perform purification of unfolded protein in urea
insufficiently exposed. or Gua-HCl as for inclusion bodies. To minimize
dilution of the sample, solid urea or Gua-HCl can
be added.
Buffer/sample composition Check pH and composition of sample and
is incorrect. binding buffer. Ensure that chelating or strong
reducing agents are not present in the sample
at too high concentration, and that the
concentration of imidazole is not too high.
Histidine tag has been lost. Check sequence of the construct on Western
blot or extract using anti-His antibody.
Incubation time is too short. Decrease the flow rate or increase the incubation
time of the sample in the wells/batch or use a
lower centrifugation speed/vacuum.
Histidine-tagged protein Elute with a larger volume of elution buffer
is not completely eluted. and/or increase the concentration of imidazole.
Histidine-tagged protein Sonication may be insufficient. Cell disruption may be checked by microscopic
found in the pellet examination or monitored by measuring the
(SDS-PAGE of samples release of nucleic acids at A260. Addition of
collected during the lysozyme (up to 0.1 volume of a 10 mg/ ml
preparation of the lysozyme solution in 25 mM Tris-HCl, pH 8.0)
bacterial lysate may prior to sonication may improve results. Avoid
indicate that most of frothing and overheating as this may denature
histidine-tagged protein the target protein. Over-sonication can also lead
is located in the to copurification of host proteins with the target
centrifugation pellet) protein.
Protein was adsorbed to cell Change extraction condition (pH, ionic strength,
debris during extraction and lost try detergent solubilization).
upon clarification
The protein may be insoluble The protein can usually be solubilized (and
(inclusion bodies). unfolded) from inclusion bodies using common
denaturants such as 4 to 6 M Gua-HCl, 4 to
8 M urea, or strong detergents. Prepare buffers
containing 20 mM sodium phosphate, 8 M urea,
or 6 M Gua-HCl, and suitable imidazole
concentrations, pH 7.4 to 7.6. Buffers with urea
should also include 500 mM NaCl. Use these
buffers for sample preparation, as binding buffer
and as elution buffer. For sample preparation
and binding buffer, use 5 to 40 mM imidazole or
the concentration selected during optimization
trials (including urea or Gua-HCl). To minimize
dilution of the sample, solid urea or Gua-HCl can
be added.
The eluted protein is not Proteases have partially degraded Add protease inhibitors (use EDTA with caution).
pure (multiple bands on the tagged protein.
SDS-PAGE)
Contaminants have high affinity Elute with a stepwise or linear imidazole
for the metal ion. gradient to determine optimal imidazole
concentrations to use for binding and for wash;
add imidazole to the sample in the same
concentration as the binding buffer. Wash before
elution with binding buffer containing as high
concentration of imidazole as possible, without
causing elution of the tagged protein.
A shallow imidazole gradient (20 column
volumes or more), may separate proteins with
similar binding strengths. If optimized conditions
do not remove contaminants, further purification
steps may be necessary.
Contaminants are associated Add detergent and/or reducing agents before
with tagged protein, sonicating cells. Increase detergent levels (e.g.,
e.g., chaperonins attached to up to 2% Triton X-100 or 2% Tween 20), or add
the target protein. glycerol (up to 50%) to the wash buffer to
disrupt nonspecific interactions.
Consider increasing the imidazole concentration
or changing the metal ion used for purification.
Unbound material has been Repeat the wash step after sample application
insufficiently removed by to obtain optimal yield.
the washing step.
Histidine-tagged protein Contaminants may have a high Charge the column using another metal ion.
is eluted during sample affinity for certain metal ions.
loading/ wash
Buffer/sample composition is Check pH and composition of sample and
not optimal. binding buffer. Ensure that chelating or strong
reducing agents are not present in the sample
at a too high concentration, and that the
concentration of imidazole is not too high.
Histidine tag is partially Purify under denaturing conditions (use 4 to
obstructed. 8 M urea or 4 to 6 M Gua-HCl).
Capacity is exceeded. For applicable formats (i.e., prepacked HisTrap
columns), join two or three columns together or
change to a larger column.
Unwanted air bubbles Unclarified lysates may cause increased air
have formed bubble formation during purification. An
attached flow restrictor in the chromatography
system can prevent this. If a flow restrictor is
attached, it is important to change the pressure
limit to 0.5 MPa (5 bar) on the ÄKTAdesign system
(where the column and the flow restrictor give a
pressure of 0.3 MPa and 0.2 MPa, respectively).
MultiTrap: Add 500 µl of deionized water twice before
Leakage of solution adding binding buffer to the wells. Remove
after removing foils the solution between the additions with either
centrifugation or vacuum.
MultiTrap: Increase/decrease the vacuum.
Problem with Add more wash steps before eluting the protein.
reproducibility Change to centrifugation.
and/or foam in
collection plate
when using
vacuum
Chapter 4
Optimizing purification of histidine-tagged
proteins
Introduction
Three methods for optimizing purification of histidine-tagged proteins are discussed in this chapter:
• Optimizing using imidazole
• Optimizing using different metal ions
• Optimizing using multistep purifications
For general purification of histidine-tagged proteins, including typical workflow, descriptions of
available media and product formats, procedures, and troubleshooting hints, refer to Chapter 3.
Optimizing using imidazole

The presence of surface-exposed histidine residues or other complex-forming amino acids can lead to
nonspecific binding of untagged host cell proteins to purification media. These untagged proteins elute
with the target protein and must subsequently be removed. Because the binding affinity of these
contaminants is often lower than that of the tagged recombinant proteins, separating the contaminants
from the protein of interest may be possible by optimizing the separation conditions.
The several examples below show how changes in imidazole concentration affect the purity of the
histidine-tagged target protein.
1. Employing imidazole as a competitive agent

In chromatographic runs that include imidazole as elution agent, the column should be preequilibrated
with a low concentration of imidazole. If this step is omitted, uncontrollable effects may occur once the
imidazole is introduced during the run.
One way to reduce the binding of contaminant proteins during purification is to employ imidazole as a
competitive agent (see Fig 45). In the experiment shown, histidine-tagged protein kinase G [(His)6-PknG]
from Mycobacterium bovis was purified using a concentration of 45 mM imidazole in the sample and
binding buffer during final purification. The medium used in the experiment was Ni Sepharose High
Performance (see Chapter 3).
To achieve a higher protein concentration, the protein was eluted in a two-step gradient (Fig 45A). To
demonstrate the advantageous effect of imidazole, an additional purification was performed under the
same conditions except that imidazole was omitted from the sample and binding buffer (Fig 45B). It is
important to note that omission of imidazole is not generally recommended; this example is provided
solely to demonstrate the negative effect of its absence on the purity of the eluted target protein.
SDS-PAGE of the pooled elution fractions indicated a large improvement in purity of the desired protein
when 45 mM imidazole was included in the sample and binding buffer (Fig 45C). The yield of the target
protein was maintained in the sample with 45 mM imidazole present. Note that the concentration of
imidazole is protein dependent and thus must be determined case by case.
A) Column: Ni Sepharose High Performance,
mAU 2 ml in XK 16/20
3500 Sample: Histidine-tagged PknG in 26 ml E. coli
M15 extract
3000 Binding buffer: 20 mM Tris, pH 8.0, 0.5 M NaCl, 1 % Triton X-100,
2500 10% glycerol, 10 mM β-mercaptoethanol,
45 mM imidazole
2000 Elution buffer: 20 mM Tris, pH 8.0, 0.5 M NaCl, 1% Triton X-100,
10% glycerol, 10 mM β-mercaptoethanol,
1500
500 mM imidazole
1000 Gradient: step 50% elution buffer, 20 CV;
100% elution buffer 20 CV
500 3–8
Flow rate: 1 ml/min
0 System: ÄKTApurifier 10
0 20 40 60 80 100 ml
B) Column: Ni Sepharose High Performance,

2 ml in XK 16/20
mAU
3500 Sample: Histidine-tagged PknG in 26 ml E. coli
M15 extract
3000 Binding buffer: 20 mM Tris, pH 8.0, 0.5 M NaCl, 1% Triton X-100,
2500 10% glycerol, 10 mM β-mercaptoethanol
Elution buffer: 20 mM Tris, pH 8.0, 0.5 M NaCl, 1% Triton X-100,
2000 10% glycerol, 10 mM β-mercaptoethanol,
500 mM imidazole
1500
Gradient: step 50% elution buffer, 20 CV;
1000 100% elution buffer 20 CV
500 Flow rate: 1 ml/min
System: ÄKTApurifier 10
0 3–8
0 20 40 60 80 100 ml
C)
Imidazole
- + M Mr
175 000
83 000
62 000
47 500
32 500
25 000
16 500
Fig 45. Purification of (His)6-PknG with (A) and without (B) 45 mM imidazole in the sample and binding buffer. For each chromatogram,
the lysate of 2 l of E. coli culture (sample volume 26 ml; filtered through a 0.45-µm syringe filter) was loaded on a 2-ml Ni Sepharose High
Performance column (XK 16/20 column) using ÄKTApurifier. The kinase was eluted in a two-step gradient with 50% and 100% of elution
buffer. (C) SDS-PAGE (12% gel) of (His)6-PknG fractions showing eluates without (-) and with (+) 45 mM imidazole in the binding buffer.
2. Determining optimal imidazole concentration using His SpinTrap
The imidazole concentration during binding and washing is an important factor affecting the final
purity and yield of the target protein. His SpinTrap is a convenient and fast tool for determination of
optimal imidazole concentration. Optimization is important for both purity and yield of the target
protein. This was demonstrated by a series of experiments where a histidine-tagged protein, APB7-(His)6
(Mr 28 000), was purified on His SpinTrap using 5, 50, 100, or 200 mM imidazole in samples and binding
buffers. The elution buffer contained 500 mM imidazole.
An imidazole concentration of 5 mM resulted in low purity of the eluted sample (Fig 46, lane 3), while
an increase to 50 mM imidazole prevented binding of most contaminants and improved purity (Fig 46,
lane 4). Including 100 mM imidazole in the sample and binding buffer lowered yield while purity improved
marginally (Fig 46, lane 5). The lower yield can be explained by leakage of target protein due to the
high imidazole concentration during binding and washing. Further increase to 200 mM imidazole
reduced yield even more (Fig 46, lane 6).
This example shows that higher imidazole concentrations during binding improve the purity, whereas
too high concentration decreases the yield. The optimal imidazole concentration during binding is
protein dependent. For many proteins, 20 to 40 mM imidazole is the best choice.
Column: His SpinTrap
Equilibration: 600 µl binding buffer
Sample application: 600 µl clarified E. coli BL-21 lysate containing 400 µg APB7-(His)6
Wash: 600 µl binding buffer
Elution: 2 × 200 µl elution buffer
Binding buffer: 20 mM sodium phosphate, 500 mM NaCl, 5–200 mM imidazole, pH 7.4
Mr
97 000
66 000
45 000
30 000 Lanes
1. LMW markers
3. Eluted pool, 5 mM imidazole during binding (diluted 1:2)
14 400
1 2 3 4 5 6 6. Eluted pool, 200 mM imidazole during binding (diluted 1:2)
Fig 46. SDS-PAGE under reducing conditions (ExcelGel SDS Gradient 8–18) of histidine-tagged APB7 protein. The imidazole concentration
during binding affects the final purity and yield (compare lanes 3, 4, 5, and 6).
Optimizing using different metal ions
The strength of binding between a protein and a metal ion is affected by several factors, including the
structure and characteristics of the target protein, the presence and properties of the protein affinity
tag, the properties of the metal ion, and the pH and composition of the binding buffer. As a result, Ni2+,
the metal ion considered to have the strongest affinity to histidine-tagged proteins, may not always be
the best choice for a given application. Under some circumstances, therefore, other transition metal
ions, such as Ca2+, Co2+, Cu2+, Fe3+, and Zn2+, may be better suited.
When the binding characteristics of a target protein are unknown, we recommend testing more than
one metal ion to determine the one best suited for your separation. GE Healthcare offers several
uncharged IMAC purification products for such purposes: convenient, prepacked 1-ml and 5-ml HiTrap
IMAC HP and HiTrap IMAC FF and 20-ml HiPrep IMAC FF 16/10 columns, as well as IMAC Sepharose
High Performance and IMAC Sepharose 6 Fast Flow bulk media. These products are described in
Chapter 3, which also includes procedures for their use.
The following guidelines may assist in devising preliminary experiments to determine the metal ion
most suitable for a given separation:
• Ni2+ is generally used for histidine-tagged recombinant proteins.
• Co2+ is also used for purification of histidine-tagged proteins, especially when a somewhat weaker
binding of target proteins is preferred.
• Cu2+ and Zn2+ are frequently used for purification of untagged proteins. Cu2+ gives relatively strong
binding to a range of proteins; some proteins will only bind to Cu2+. Zn2+ ions often bind more weakly, a
characteristic that is often exploited to achieve selective elution of the target protein. Both Cu2+ and
Zn2+ can be used for histidine-tagged proteins and for process-scale separations.
• Fe3+ and Ca2+ are used more rarely than other metal ions. Take extra precautions when working with
Fe3+, as it reduces easily in neutral solutions, forming compounds that can be hard to dissolve. When
working with Fe3+ at low pH, approximately 3, immobilize the ions to avoid precipitation of unwanted
compounds. We also advise stripping immobilized Fe3+ ions after each run and recharging the column
as required. Strongly bound Fe3+ ions and ferric compounds can be removed by leaving the medium
in 50 mM EDTA overnight.
Below we present two examples showing how the selection of the most suitable metal ion and
experimental conditions (including imidazole concentration) affects the purification of a given target
protein.
1. Comparison study using Cu2+, Zn2+, and Ni2+ on HiTrap IMAC FF

In this study, APB7, a (histidine)6-tagged protein (Mr 28 000) expressed in E. coli BL-21, was purified on
HiTrap IMAC FF 1-ml columns (prepacked with IMAC Sepharose 6 Fast Flow) and charged separately
with Cu2+, Zn2+, and Ni2+.
Screening experiments were performed to determine the optimal imidazole concentration for each ion
[see Figure 47 (A to C)]. The results of each of these three purifications indicate:
• At 20 mM imidazole, there was significant leakage of target protein in the wash with Cu2+ and Zn2+, a
sign that imidazole concentration was too high to allow maximal yield. Very little leakage was seen
with Ni2+. Purity was excellent in all three cases.
• At 10 mM imidazole, leakage of target protein in the wash was significantly reduced for Cu2+ and Zn2+.
The purity of the target protein in the eluted pool was similar with all three metal ions, but not as
pure as with 20 mM imidazole.
• At 5 mM imidazole, no leakage occurred with any of the metal ions. Ni2+ provided the purest protein,
though still not as pure as with 20 mM imidazole.

A) 20 mM imidazole Lanes
Cu2+ Zn 2+ Ni2+
1. LMW markers
Mr 2. Start material APB7, diluted 1:10
66 000 4. Wash, Cu2+
45 000 5. Eluted pool, Cu2+
30 000 6. Flowthrough, diluted 1:10, Zn2+
20 100 7. Wash, Zn2+
14 400 8. Eluted pool, Zn2+
1 2 3 4 5 6 7 8 9 10 11 9. Flowthrough, diluted 1:10, Ni2+
10. Wash, Ni2+
B) 10 mM imidazole Lanes
Cu2+ Zn2+ Ni2+ 1. LMW markers
66 000 4. Wash, Cu
2+

2+
20 100 7. Wash, Zn
9. Flowthrough, diluted 1:10, Ni2+
1 2 3 4 5 6 7 8 9 10 11 2+
10. Wash, Ni
C) 5 mM imidazole Lanes
Cu2+ Zn2+ Ni2+
1. LMW markers
66 000 2+
4. Wash, Cu
7. Wash, Zn2+
20 100
9. Flowthrough, diluted 1:10, Ni2+
1 2 3 4 5 6 7 8 9 10 11 10. Wash, Ni2+
Fig 47. SDS-PAGE analyses (reducing conditions) of fractions from the purification of APB7 using IMAC Sepharose 6 Fast Flow, prepacked in
HiTrap IMAC FF 1-ml columns, charged with either Cu2+, Zn2+, or Ni2+, and with (A) 20 mM imidazole in the sample, (B) 10 mM imidazole in
the sample, or (C) 5 mM imidazole in the sample. The gels were stained with Coomassie.
Note that a large amount of sample was applied in the SDS-polyacrylamide gel. Because of this, the
gels show a number of contaminants in the eluted material.
The results illustrate that, for any given metal ion, imidazole concentration can be adjusted to achieve
high yield, high purity, or a successful compromise. IMAC Sepharose media typically requires a slightly
higher concentration of imidazole in the wash buffer than similar IMAC media on the market . A good
starting point for most separations is to include 20 to 40 mM imidazole in the binding and wash buffers.
Be sure to use highly pure imidazole, which gives essentially no absorbance at 280 nm. To remove
imidazole from the protein, use HiTrap Desalting, PD-10 Desalting, or HiPrep 26/10 Desalting column,
depending on the sample volume.

2. Screening for optimized purity using Cu2+, Zn2+, Co2+, and Ni2+ on HiTrap IMAC HP
Successful purifications require attention to the properties of the target protein and the affinity of the
target protein toward the metal ion used. Four different HiTrap IMAC HP 1-ml columns prepacked with
IMAC Sepharose High Performance were charged separately with four different metal ions: Cu2+, Zn2+,
Co2+, and Ni2+. Purification performance was assessed for the target protein: APB7, (Mr 28 000)
expressed in E. coli.
The results demonstrate the importance of screening samples to determine the most suitable metal
ion and purification conditions for specific target proteins. For APB7, the highest purity was achieved
using Ni2+ or Co2+, but the difference compared to the results for Zn2+ and Cu2+ was small (Fig 48). The
gradient elutions with imidazole employed in these examples also offer a methodology for selecting
the appropriate imidazole concentration for a given purification.
mAU %B Columns: HiTrap IMAC HP, 1 ml, charged with:

A) Cu2+ A) Cu2+ B) Zn2+ C) Co2+ D) Ni2+
800 80 Conditions were otherwise the same for the
four purifications
600 60 Sample: E. coli extract with APB7, a (histidine)6-tagged
protein (Mr ~28 000), including 30 mM imidazole
400 40 Sample volume: 10 ml
0 0
500 mM imidazole, pH 7.4
Cu2+
0.0 10.0 20.0 30.0 40.0 50.0 60.0 ml

Flow rate: 1 ml/min
pool Gradient: 6–60% elution buffer (30–300 mM imidazole)
mAU %B
in 25 m 100% elution buffer (500 mM imidazole)
B) Zn2+
800 80 in 5 ml
Detection: Absorbance, 280 nm
400 40
200 20
0 0 E)
0.0 10.0 20.0 30.0 40.0 50.0 60.0 ml
Zn 2+
mAU pool %B Mr
C) Co2+ 97 000
800 80 66 000
45 000
600 60
30 000
400 40 20 100
14 400
200 20
1 2 3 4 5 6 7 8 9 10 11 12
0 0
0.0 10.0 20.0 30.0 40.0 50.0 60.0 ml
Co2+
Lanes
1. LMW markers
mAU pool %B
2. Start material, diluted 1:10
D) Ni2+ 3. Flowthrough, diluted 1:10, Cu
2+
800 80
4. Wash, Cu2+
2+
6. Wash, Zn
8. Wash, Co2+
200 20 2+
9. Eluted pool, Co
0 0 10. Wash, Ni2+
0.0 10.0 20.0 30.0 40.0 50.0 60.0 ml 11. Eluted pool, Ni2+
Ni 2+
pool 12. LMW markers
Fig 48. Purification of APB7, a (histidine)6-tagged protein expressed in E. coli BL-21 on four different HiTrap IMAC HP 1-ml columns
charged separately with metal ions. (A) Cu2+, (B) Zn2+, (C) Co2+, or (D) Ni2+. Pools selected after SDS-PAGE of individual 1-ml fractions (not
shown) are indicated. (E) SDS-PAGE analysis: reducing conditions on ExcelGel SDS Gradient 8–18; Coomassie staining.

Optimizing using multistep purifications
Target protein can be further purified by adding one or more additional purification steps, as shown in
the examples below. This topic is discussed in detail in Chapter 7.
Below we present two examples showing successful multistep purification of the target proteins.
1. Two-step purification of a high-molecular-weight (histidine)10-tagged protein using

affinity chromatography and gel filtration
In a two-step purification of (His)10-trx-p450 (histidine-tagged at the N-terminus, 10 histidine residues)
produced in E. coli, a HisTrap FF column was used in the first step. The eluted pool was then applied to
a HiLoad 16/60 Superdex 200 pg column for further purification by gel filtration.
Three major bands were detected after the first purification step (Fig 49B, lane 5). Lane 6 (pool 2 from
gel filtration) contains full-length target protein. Lanes 7 and 8 (pools 3 and 4, respectively) contain
truncated forms of the target protein, as verified by N-terminal sequencing (data not shown). Lane 9
(pool 1) from gel filtration contains aggregated proteins. Thus subsequent gel filtration provided very
good separation between the truncated forms and the full-length target protein, (His)10-trx-p450.
Columns: Affinity chromatography (AC): HisTrap FF 1-ml

Gel filtration (GF): HiLoad 16/60 Superdex 200 pg
Sample: E. coli extract with (His)10-trx-p450, (Mr ~130 000)
Binding buffer: AC: 20 mM sodium phosphate, 60 mM imidazole, 500 mM NaCl, pH 7.4
Elution buffer: AC: 20 mM sodium phosphate, 500 mM imidazole, 500 mM NaCl, pH 7.4
GF: 20 mM phosphate, 0.28 M NaCl, 6 mM KCl, pH 7.4
Flow rate: AC: 1 ml/min
GF: 0.5 ml/min
A)
A280 HisTrap FF 1-ml A 280 HiLoad 16/60 Superdex 200 pg
mAU mAU
400
4000
300
3000
200
2000 Pool
100
1000
Pool 4
Pool 2
Pool 3
0
Pool 1
Pool
0
0.0 20.0 40.0 60.0 80.0 ml 0 50 100 ml
B) Lanes
Mr 1. LMW markers
97 000 2. Start material, 1:30
66 000 3. Flowthrough AC, 1:20
45 000 4. Wash AC, 1:10
5. Pool AC, 1:10
30 000 6. Pool 2, GF, 1:2
20 100 7. Pool 3, GF, 1:2
14 400 8. Pool 4, GF, 1:2
1 2 3 4 5 6 7 8 9 9. Pool 1, GF
Fig 49. (A) Two-step purification of a high-molecular-weight (histidine)10-tagged protein using affinity chromatography followed by gel
filtration. (B) SDS-PAGE under reducing conditions and Coomassie staining.

2. Automatic three-step purification of unclarified cell lysate on ÄKTAxpress
An automated three-step protocol was used to purify histidine-tagged maltose binding protein from
100 ml of an unclarified E. coli cell lysate. The three steps were: Affinity chromatography (AC) using
HisTrap FF crude (1-ml column), desalting (DS) using HiPrep 26/10 Desalting, and ion exchange
chromatography (IEX) using Mono Q™ 5/50 GL. These are referred to as AC-DS-IEX in the image. As can
be seen from the SDS-PAGE analysis, the target protein was obtained highly pure and in good yield.
Columns: Affinity chromatography (AC): HisTrap FF crude, 1 ml

Desalting (DS): HiPrep 26/10 Desalting
Ion exchange (IEX): Mono Q 5/50 GL
Sample: Histidine-tagged Maltose Binding Protein, MBP-(His)6, Mr 43 000, in E. coli DH5α extract
AC binding buffer: 50 mM Tris-HCl, 0.5 M NaCl, 20 mM imidazole, pH 8.0
AC elution buffer: 50 mM Tris-HCl, 0.5 M NaCl, 500 mM imidazole, pH 8.0
DS/IEX binding buffer: 50 mM Tris-HCl, pH 8.0
IEX elution buffer: 50 mM Tris-HCl, 1.0 M NaCl, pH 8.0
A)
mAU
2000 AC mAU
1500
IEX
1000
IEX
1000 DS 800
500 600
0 400
0 50 100 150 200 ml
200
B) 0
170.0 180.0
Mr Pool 1 2 3
97 000
66 000
45 000
30 000 Lanes
20 100 1. LMW markers
2. Start material, 1:10
14 400
3. Eluted pool 1 from IEX
4. Eluted pool 2 from IEX
1 2 3 4 5 5. Eluted pool 3 from IEX
Fig 50. (A) AC-DS-IEX with an enlargement of the IEX peaks and the collected pools to the right. Yield: 9.4 mg in pools 1 + 2. (B) SDS-PAGE
of eluted pools from IEX. The gel was stained with Coomassie.

Chapter 5
Purification of GST-tagged recombinant proteins
Introduction
Use of the Glutathione S-transferase (GST) affinity tag was first introduced in 1988 and has since
become a popular choice when working with recombinant proteins. The method is based on the affinity
of GST to the glutathione ligand coupled to a matrix. The binding of a GST-tagged protein to the ligand
is reversible, and the protein can be eluted under mild, nondenaturing conditions by the addition of
reduced glutathione to the elution buffer. The technique thus provides a purification process that
preserves protein antigenicity and function.
The GST Gene Fusion System is a versatile system from GE Healthcare for the expression, purification,
and detection of GST-tagged proteins produced in E. coli. The system consists of three major components:
pGEX plasmid vectors, products for GST purification, and a GST detection kit. A series of site-specific
proteases complements the system.
GST occurs naturally in most organisms. Tagged proteins that possess the complete amino acid
sequence of GST also demonstrate GST enzymatic activity and can undergo dimerization similar to
that observed in nature. The crystal structure of recombinant Schistosoma japonicum GST (Mr 26 000)
from pGEX vectors has been determined.
The pGEX vectors are designed for inducible, high-level intracellular expression of genes or gene
fragments as fusions with S. japonicum GST. Expression in E. coli yields tagged proteins with the GST
moiety at the amino terminus and the protein of interest at the carboxyl terminus.
A variety of affinity chromatography products are available from GE Healthcare that have glutathione
immobilized to one of three Sepharose media: Sepharose High Performance (HP), Sepharose 6 Fast
Flow (FF), or Sepharose 4B. The Glutathione Sepharose media are available in several formats, ranging
from 96-well filter plates to prepacked HiTrap and HiPrep columns to lab packs (media packs in sizes
from 25 ml to 500 ml). The media vary in their performance parameters, and the different formats
provide options of scale and convenience.
If desired, cleavage of the protein from GST can be achieved using a site-specific protease whose
recognition sequence is located immediately upstream from the multiple cloning site on the pGEX
plasmids. Tagged proteins can be detected using colorimetric or immunological methods. Cleavage
and detection options are discussed later in this chapter.
This chapter summarizes key aspects of working with GST-tagged proteins, with a focus on purification
methodologies. For more detailed information on the GST system, refer to the GST Gene Fusion System
Handbook (Code No. 18-1157-58); the handbook includes detailed information on expression, purification,
detection, and removal of the GST tag and is an invaluable guide when working with the system.
Expression
Selecting an expression strategy begins with choosing the vector best-suited for your purpose, taking
note of reading frame, cloning sites, and protease cleavage sites. Correct preparation of the insert is
important and must take into account the reading frame and orientation, size, and compatibility of the
fragment ends. Selection of host cells involves consideration of cloning and maintenance issues and
anticipated expression levels. Finally, growth conditions must be evaluated in order to optimize
expression. These topics are discussed below.

pGEX vectors
GST-tagged proteins are constructed by inserting a gene or gene fragment into the multiple cloning
site of one of the pGEX vectors. Expression is under the control of the tac promoter, which is induced
by the lactose analog isopropyl β-D thiogalactoside (IPTG). All pGEX vectors are also engineered with
an internal lacIq gene. The lacIq gene product is a repressor protein that binds to the operator region of
the tac promoter, preventing expression until induction by IPTG, thus maintaining tight control over
expression of the insert.
Because of the mild elution conditions for release of tagged proteins from the affinity medium, effects
on antigenicity and functional activity of the protein are minimized. The vectors have a range of
protease cleavage recognition sites as shown in Table 15.
Table 15. Protease cleavage sites of pGEX vectors.
Vector Cleaved by
pGEX-6P-1, pGEX-6P-2, pGEX-6P-3 PreScission Protease
pGEX-4T-1, pGEX-4T-2, pGEX-4T-3 Thrombin
pGEX-5X-1, pGEX-5X-2, pGEX-5X-3 Factor Xa
pGEX-2TK Thrombin
Allows detection of expressed proteins
by direct labeling in vitro
The vectors provide all three translational reading frames beginning with the EcoR I restriction site (see
Appendix 7). The same multiple cloning sites in each vector ensure easy transfer of inserts. pGEX-6P-1,
pGEX-4T-1, and pGEX-5X-1 can directly accept and express cDNA inserts isolated from λgt11 libraries.
pGEX-2TK has a different multiple cloning site from that of the other vectors. pGEX-2TK is uniquely
designed to allow the detection of expressed proteins by directly labeling the tagged products in vitro.
This vector contains the recognition sequence for the catalytic subunit of cAMP-dependent protein
kinase obtained from heart muscle. The protein kinase site is located between the thrombin recognition
site and the multiple cloning site. Expressed proteins can be directly labeled using protein kinase and
[γ-32P]ATP and readily detected using standard radiometric or autoradiographic techniques.
Refer to Appendix 7 for a listing of the control regions of the pGEX vectors. Complete DNA sequences and
restriction site data are available with each individual vector’s product information, at the GE Healthcare
Web site (http://www.gehealthcare.com/lifesciences) and also from GenBank™. GenBank accession
numbers are listed in Appendix 7.
Select the proper vector to match the reading frame of the cloned insert.
Consider which protease and conditions for cleavage are most suitable for your target protein
preparation.
pGEX-6P PreScission Protease vectors offer the most efficient method for cleavage and purification of
GST-tagged proteins. Site-specific cleavage may be performed with simultaneous immobilization of
the protease on the column. The protease has high activity at low temperature so that all steps can be
performed in the cold room to protect the integrity of the target protein. Cleavage enzyme and GST
tag are removed in a single step, as described later in this chapter.

The host
Although a wide variety of E. coli host strains can be used for cloning and expression with the pGEX
vectors, there are specially engineered strains that are more suitable and that may maximize expression
of full-length tagged proteins. Strains deficient in known cytoplasmic protease gene products, such as
Lon, OmpT, DegP or HtpR, may aid in the expression of tagged proteins by minimizing the effects of
proteolytic degradation by the host.
A lyophilized (noncompetent) culture of E. coli BL21 is supplied with all pGEX vectors and is also
available separately.
Using E. coli strains that are not protease-deficient may result in proteolysis of the tagged protein,
seen as multiple bands on SDS-PAGE or Western blots.
E. coli BL21, a strain defective in OmpT and Lon protease production, gives high levels of expression of
GST-tagged proteins. It is the host of choice for expression studies with GST-tagged proteins.
Use an alternative strain for cloning and maintenance of the vector (e.g., JM105) because BL21
does not transform well. However, do not use an E. coli strain carrying the recA1 allele for
propagation of pGEX plasmids. There have been reports that these strains can cause
rearrangements or deletions within plasmid DNA.
Insert DNA
Insert DNA must possess an open reading frame and should be less than 2 kb long. Whether subcloned
from another vector or amplified by PCR, the insert must have ends that are compatible with the
linearized vector ends. Using two different restriction enzymes will allow for directional cloning of the
insert into the vector. Directional cloning will optimize for inserts in the correct orientation.
Optimizing expression
Once it has been established that the insert is in the proper orientation and that the correct junctions
are present, the next step is to optimize expression of tagged proteins. The capability to screen crude
lysates from many clones is critical to this process, so that optimal expression levels and growth
conditions can be readily determined. Once conditions are established, one is ready to prepare large-
scale bacterial sonicates of the desired clones.
To screen many putative clones simultaneously, several purification methods are recommended. The
first method uses GST MultiTrap FF or GST MultiTrap 4B 96-well plates, which are designed to allow
parallel purification of GST-tagged proteins directly from unclarified cell lysates (maximum 600 µl per
well). In the second method, a crude lysate suitable for screening from 2 to 3 ml of culture is prepared,
using a batch purification method with one of the Glutathione Sepharose media. In the third method,
the GST SpinTrap Purification Module is used. This module can isolate protein from up to 12 ml of
culture using a standard microcentrifuge. All of these methods are presented later in this chapter.
In addition, several options are presented later in this chapter for determining expression levels.
Growth conditions should be evaluated for optimal expression: media, growth temperature, culture
density, induction conditions, and other variables should be evaluated. It is important to assure sufficient
aeration and to minimize the time spent in each stage of growth, as well as to use positive selection
for the plasmid (antibiotic resistance). Formation of inclusion bodies should be monitored and possibly
be avoided by optimizing expression. This topic is discussed in Chapter 8.
Monitor both cell density (A600) and protein expression for each variable evaluated.

Purification
GST-tagged proteins are easily purified from bacterial lysates by affinity chromatography using
glutathione immobilized to a matrix such as Sepharose (Fig 51). When applied to the affinity medium,
tagged proteins bind to the ligand, and impurities are removed by washing with binding buffer. Tagged
proteins are then eluted from the Glutathione Sepharose under mild, nondenaturing conditions that
preserve both protein antigenicity and function.
If separation of the cloned protein from the GST affinity tag is desired, the tagged protein can be
digested with an appropriate site-specific protease while the protein is bound to Glutathione
Sepharose. Alternatively, the tagged protein can be digested following elution from the medium (see
later in this chapter for both of these alternatives). Cleavage of the bound tagged protein eliminates
the extra step of separating the released protein from GST because the GST moiety remains bound to
the medium while the cloned protein is eluted using wash buffer.
O O
C
H N
C O
CH2 CH2 S
O C N H
H OH O C
NH3 +
C
O O
Fig 51. Terminal structure of Glutathione Sepharose. Glutathione is specifically and stably coupled to Sepharose by reaction of the
SH-group with oxirane groups obtained by epoxy-activation of the Sepharose matrix. The structure of glutathione is complementary
to the binding site of glutathione S-transferase.

Selecting a product for GST-tagged protein purification
Products designed to meet specific purification needs are available for purification of GST-tagged proteins,
as shown in the selection guide in Table 16. These products rely on affinity chromatography using
gravity flow, centrifugation, vacuum, syringe, or pump action to purify the protein. A comparison of the
physical characteristics of the three Glutathione Sepharose media is given in Appendix 2.
Table 16. Selection guide summarizing purification options for GST-tagged proteins, including companion products.
Product Plate/column Plate/column Description Application

size capacity
Glutathione 25 ml Approx. 10 mg Lab pack For high resolution and
Sepharose High 100 ml (rGST) elution of a more
Performance concentrated sample (high-
performance purification).
Glutathione 25 ml Approx. 10 mg Lab pack Excellent for scale-up due to
Sepharose 4 100 ml (rGST) good binding capacity and
Fast Flow 500 ml good flow properties.
Glutathione 10 ml > 5 mg Lab pack Good binding capacity.
Sepharose 4B 100 ml (rGST)
(function
tested)
300 ml
Glutathione 2 ml Up to 10 mg 2 columns prepacked with Gravity flow.
Sepharose 4B (horse liver GST) Glutathione Sepharose 4B. No system needed.
GST MultiTrap FF 50 µl Up to 0.5 mg Prepacked 96-well filter For high-throughput
(rGST) plates with Glutathione 4 expression screening.
Fast Flow. For use with robotics or
manually by centrifugation
or vacuum.
GST MultiTrap 4B 50 µl Up to 0.5 mg Prepacked 96-well filter For high-throughput
(rGST) plates with Glutathione expression screening.
Sepharose 4B. For use with robotics or
manually by centrifugation
or vacuum.
GST SpinTrap 50 µl Up to 0.4 mg 50 columns and reagents. For use in a microcentrifuge.
Purification (rGST) Columns prepacked with Medium throughput for
Module Glutathione Sepharose 4B. expression screening and
minipreps.
GSTrap HP 1 ml Approx. 10 mg Columns prepacked with For use with a peristaltic
5 ml Approx. 50 mg Glutathione Sepharose pump or chromatography
(rGST) High Performance. system in preference over
syringe.
For high resolution and elution
of a more concentrated
sample (high-performance
purification).
GSTrap FF 1 ml Approx. 10 mg Columns prepacked with For use with syringe,
5 ml Approx. 50 mg Glutathione Sepharose 4 peristaltic pump, or
(rGST) Fast Flow. chromatography system.
Provides good flow properties.
Scale-up.
GSTrap 4B 1 ml > 5 mg Columns prepacked with For use with syringe,
5 ml > 25 mg Glutathione Sepharose 4B. peristaltic pump or
(horse liver GST) chromatography system.
GSTPrep™ FF 20 ml Approx. 200 mg Columns prepacked with For use with a
16/10 (rGST) Glutathione Sepharose chromatography system.
4 Fast Flow. Scale-up purification.

Table 16. Selection guide summarizing purification options for GST-tagged proteins, including companion products (continued).
Product Pack Capacity Description Application

size
pGEX Vectors 5 to 25 µg N/A Vector and E. coli BL21 A tac promoter for chemically
(GST Gene Fusion vector cells. inducible, high-level
System) expression. PreScission,
thrombin, or Factor Xa
protease recognition sites.
Anti-GST Antibody 0.5 ml 50 detections Anti-GST antibody. Polyclonal.
For sensitive and specific
detection of GST-tagged
proteins. For use with an
enzyme-conjugated
anti-goat antibody.
Anti-GST HRP 75 µl 1:5000 dilution, Highly specific antibody Polyclonal.
Conjugate typical to GST conjugated to HRP Offers speed, sensitivity, and
concentration and optimized for use in safety for detection of GST-
Western blotting with ECL tagged proteins. Recognizes
detection reagents. multiple epitopes of GST,
thus not reliant on functional
GST for detection.
GST 96-Well 5 plates N/A GST 96-Well Detection Plates precoated with Anti-
Detection Module HRP Module. GST antibody and blocked
conjugated for the capture of GST-
Anti-GST tagged proteins, which are
Antibody then detected using HRP
and conjugated Anti-GST
GST protein. Antibody.
GST Detection 1-chloro-2 50 detection GST Detection Module. For the biochemical or
Module -4-dinitro- reactions immunological detection of
benzene GST-tagged proteins.
(CDNB), Glutathione and CDNB serve
Anti-GST as substrates to yield a yellow
Antibody, and product detectable at 340 nm.
instructions. The antibody is suitable for
use in Western blots.
ECL GST Western For 1000 or ECL Plus Solution A Acridan-based substrate. For chemiluminescent and
Blotting Detection 3000 cm2 and ECL Plus chemifluorescent detection.
Kit membrane Solution B Extended signal duration
allows multiple exposures to
be made.
PreScission 500 units One unit cleaves PreScission Protease. For specific, low-temperature
Protease > 90% of 100 µg of cleavage between Gln and
a test GST-tagged Gly residues in the sequence
protein when Leu-Glu-Val-Leu-Phe-Gln-
incubated in Gly-Pro. A tagged protein
1 mM EDTA, consisting of human
1 mM DTT, rhinovirus protease and GST.
150 mM NaCl, and Can be used for tag cleavage
50 mM Tris-HCl when the PreScission
(pH 7.0) at 5°C protease recognition
for 16 h. sequence occurs between
the tag sequence and the
protein of interest, e.g., the
GST tag from proteins
expressed using the pGEX-6P
vector.

Table 16. Selection guide summarizing purification options for GST-tagged proteins, including companion products (continued).
Product Pack Capacity Description Application

size
Thrombin 500 units One unit cleaves Thrombin For specific cleavage at the
> 90% of 100 µg recognition sequence for
of a test GST-tagged thrombin. Can be used for
protein when tag cleavage when the
incubated in 1x PBS thrombin recognition
at 22°C for 16 h. sequence occurs between
the tag sequence and the
protein of interest, e.g., the
GST tag from proteins
expressed using the pGEX-T
vectors.
Factor Xa 400 units One unit cleaves Factor Xa For specific cleavage
> 90% of 100 µg following the tetrapeptide
of a test GST-tagged Ile-Glu-Gly-Arg. Can be used
protein when for tag cleavage when the
incubated in Factor Xa recognition
1 mM CaCl2, sequence occurs between
100 mM NaCl, and the tag sequence and the
50 mM Tris-HCl protein of interest, e.g., the
(pH 8.0) at 22°C GST tag from proteins
for 16 h. expressed using pGEX-X
vectors.
HiTrap 1 ml > 35 mg trypsin Columns prepacked with Removal of serine proteases,
Benzamidine FF 5 ml > 175 mg trypsin Benzamidine Sepharose 4 e.g., thrombin and Factor Xa
(high sub) Fast Flow (high sub). after tag cleavage.
Benzamidine 25 ml > 35 mg trypsin/ml Lab pack Removal of serine proteases,
Sepharose 4 medium e.g., thrombin and Factor Xa
Fast Flow after tag cleavage.
(high sub)
Collection Plate 96-well 500 µl V-shaped bottom For use with the GST
plates MultiTrap products.

General considerations for purification of
GST-tagged proteins
Yield of tagged protein is highly variable and is affected by the nature of the tagged protein, the host
cell, and the expression and purification conditions used. Tagged protein yields can range from 1 mg/l
up to 10 mg/l. Table 17 can be used to approximate culture volumes based on an average yield
of 2.5 mg/l.
Table 17. Reagent volume requirements for different protein yields.
Tagged protein yield 50 mg 10 mg 1 mg 50 µg

Culture volume 20 l 4l 400 ml 20 ml
Volume of extract 1l 200 ml 20 ml 1 ml
Glutathione Sepharose bed volume 10 ml 2 ml 200 µl 10 µl
1× PBS1 100 ml 20 ml 2 ml 100 µl
Glutathione elution buffer 10 ml 2 ml 200 µl 10 µl
1 This volume is per wash. Three washes are required per sample in the following procedures.
Use deionized (or double-distilled) water and chemicals for sample and buffer preparation. Samples
should be centrifuged immediately before use and/or filtered through a 0.45 µm filter. If the sample is
too viscous, to prevent it from clogging the column dilute it with binding buffer, increase lysis treatment
(sonication, homogenization), or add DNase/RNase to reduce the size of nucleic acid fragments.
One of the most important parameters affecting the binding of GST-tagged proteins to
Glutathione Sepharose is the flow rate. Because the binding kinetics between glutathione and
GST are relatively slow, it is important to keep the flow rate low during sample application to
achieve maximum binding capacity. Washing and elution can be performed at a slightly higher
flow rate to save time. For batch purification, incubation time should be considered.
The binding properties of the target protein can be improved by adjusting the sample to the composition
of the binding buffer. Dilute in binding buffer or perform a buffer exchange using a desalting column such
as HiTrap Desalting 5 ml, PD-10 Desalting, or HiPrep 26/10 Desalting. Refer to Chapter 9 for use of
desalting columns.
Volumes and times used for elution may vary among tagged proteins. Further elution with higher concen-
trations of glutathione (20 to 50 mM) may improve yield. At concentrations above 15 mM glutathione,
the buffer concentration should also be increased to maintain the pH within the range 6.5 to 8.
Flowthrough, wash, and eluted material from the column should be monitored for GST-tagged proteins
using SDS-PAGE in combination with Western blot if necessary.
Following the elution steps, a significant amount of tagged protein may remain bound to the medium.
Volumes and times used for elution may vary among tagged proteins. Additional elutions may be
required. Eluates should be monitored for GST-tagged protein by SDS-PAGE or by 1-chloro-2,4
dinitrobenzene (CDNB) assay (see later in this chapter).
If monomers are desired, the GST tag should be cleaved off. Gel filtration will probably give an
unstable preparation of monomers that will start to form dimers immediately.
Batch preparation procedures are frequently mentioned in the literature. However, the availability
of prepacked columns and easily packed Glutathione Sepharose media provides faster, more
convenient alternatives. Batch preparations are occasionally used if it appears that the GST tag
is not fully accessible or when the concentration of protein in the bacterial lysate is very low
(both could appear to give a low yield from the affinity purification step). A more convenient
alternative to improve yield is to decrease the flow rate or pass the sample through the column
several times (recirculation).

Purification steps should be monitored using one or more of the detection methods described later in
this chapter. The GST Detection Module contains components that can be used for either enzymatic or
immunochemical determination of concentrations of GST-tagged proteins in extracts as well as sample
obtained during purification.
The yield of protein in purified samples can also be determined by standard chromogenic methods
(e.g., Lowry, BCA, Bradford, etc.). If a Lowry or BCA type method is to be used, the glutathione in the
purified material must be removed using, for example, HiTrap Desalting 5 ml or dialysis against 2000
volumes of PBS to reduce interference with the assay. The Bradford method can be performed in the
presence of glutathione.
Reuse of purification columns and affinity media depends upon the nature of the sample and
should only be performed with identical samples to prevent cross-contamination.
Selecting equipment for purification

The choice of equipment will depend on the specific purification. Many purification steps can be carried
out using simple methods and equipment as, for example, step-gradient elution using a syringe in
combination with prepacked HiTrap columns. Linear gradients may improve purity when GST-tagged
proteins are purified from eukaryotic hosts because endogenous GST may be co-eluted in step-gradient
elution. If the same column is to be used for many runs in series, it is wise to use a dedicated system.
Table 8 in Chapter 2 provides a guide to aid in selecting the correct purification system.
For small-scale purifications or for high-throughput screening, we recommend GST MultiTrap FF
or GST MultiTrap 4B 96-well filter plates, which can purify up to approximately 0.5 mg of GST-
tagged protein per well. In addition, GST SpinTrap columns, each containing 50 µl of Glutathione
Sepharose 4B, can purify up to 400 µg of recombinant GST.
For purification of larger quantities of GST-tagged proteins, prepacked columns such as GSTrap
and GSTPrep FF 16/10 provide excellent formats. To increase capacity, use several GSTrap
columns (1 ml or 5 ml) or two GSTPrep FF 16/10 columns (20 ml) in series or, for even larger
capacity requirements, pack Glutathione Sepharose media into a suitable column.
For simple and rapid, one-step reproducible purification, use of a chromatography system such as
ÄKTAprime plus is advantageous because it has preprogrammed methods for the most typical
applications, including purification of histidine- and GST-tagged proteins. A UV and conductivity monitor
and easy-to-use software enable automatic tracking of the protein. Monitoring is continuous in real
time, thus eliminating manual errors. For laboratory environments in which all experimental data must
be recorded and traceable, or where multistep purification schemes, method development, optimization,
and/or scale-up are needed, ÄKTApurifier or ÄKTAexplorer chromatography system is recommended.

Purification using Glutathione Sepharose High
Performance, Glutathione Sepharose 4 Fast Flow,
and Glutathione Sepharose 4B
These three media are all used for the purification of GST-tagged recombinant proteins and other
S-tranferases or glutathione-dependent proteins. They allow mild elution conditions that preserve
protein antigenicity and function. All are supplied preswollen in 20% ethanol and are also available in
various prepacked formats, such as GSTrap, as described later in this chapter. See Appendix 2 for the
main characteristics of all Glutathione Sepharose media.
In Glutathione Sepharose High Performance, the glutathione ligand is coupled to highly cross-linked
6% agarose. The medium has an average bead size of 34 µm and is an excellent choice for high-
resolution purification and elution of a more concentrated sample.
In Glutathione Sepharose 4 Fast Flow, the glutathione ligand is coupled to highly cross-linked 4% agarose.
The medium has an average bead size of 90 µm. It is a very good choice for scale-up due to its good
binding capacity and flow properties. This medium is also a good choice for batch and gravity-flow
purifications.
In Glutathione Sepharose 4B, the glutathione ligand is coupled to 4% agarose. The medium has an
average bead size of 90 µm. It provides good binding capacity, and is suitable for small-scale purification
as well as batch and gravity-flow operations.
Glutathione Sepharose 4 Fast Flow and Glutathione Sepharose 4B are also available prepacked in
96-well filter plates (see page 120).
Procedures for both batch and column purification of GST-tagged proteins follow.
Fig 52. Glutathione Sepharose High Performance and Glutathione Sepharose 4 Fast Flow for purification of GST-tagged proteins.

Batch purification of GST-tagged proteins using Glutathione Sepharose HP,
Glutathione Sepharose 4 FF, or Glutathione Sepharose 4B
Refer to page 112, General considerations, before beginning this procedure.
Sample preparation
1. Prepare the cell lysate.
2. Centrifuge the cell lysate at high speed for 10 min at 4°C and filter through a 0.45-µm filter before
applying to the Glutathione Sepharose medium. If the sample is too viscous, dilute it with binding buffer,
increase lysis treatment (sonication, homogenization), or add DNase/RNase to reduce the size of nucleic
acid fragments.
Buffer preparation
Use high-purity water and chemicals, and filter all buffers through a 0.45 µm filter before use.
Binding buffer: PBS (140 mM NaCl, 2.7 mM KCl, 10 mM Na2HPO4, 1.8 mM KH2PO4), pH 7.3
Elution buffer: 50 mM Tris-HCl, 10 mM reduced glutathione, pH 8.0
1 to 20 mM DTT may be included in the binding and elution buffers to increase the purity.
However, this may result in lower yield of GST-tagged protein.
Preparation of Glutathione Sepharose media for use in batch purification

Glutathione Sepharose media are supplied preswollen in 20% ethanol. The media are used at a final
slurry concentration of 50%.
1. Determine the bed volume of Glutathione Sepharose medium required for your purification.
2. Gently shake the bottle to resuspend the slurry.
3. Use a pipette or measuring cylinder to remove sufficient slurry for use and transfer to an appropriate
container/tube.
4. Sediment the medium by centrifugation at 500 × g for 5 min. Carefully decant the supernatant.
5. Wash the Glutathione Sepharose HP, FF, or 4B media by adding 5 ml of PBS per 1 ml of slurry (= 50% slurry).
Glutathione Sepharose media must be thoroughly washed with PBS to remove the ethanol
storage solution because residual ethanol may interfere with subsequent procedures.
7. Repeat steps 5 and 6 once for a total of two washes.
For cleaning, storage, and handling information, refer to Appendix 2.
Batch purification
1. Add the cell lysate to the prepared Glutathione Sepharose medium and incubate for at least 30 min at
room temperature, using gentle agitation such as end-over-end rotation.
2. Use a pipette or cylinder to transfer the mixture to an appropriate container/tube.
3. Sediment the medium by centrifugation at 500 × g for 5 min. Carefully decant the supernatant
(= flowthrough) and save it for SDS-PAGE analysis to determine the binding efficiency of the GST-tagged
protein to the medium.
4. Wash the Glutathione Sepharose medium by adding 5 ml of PBS per 1 ml of slurry (= 50% slurry). Invert
to mix.
5. Sediment the medium by centrifugation at 500 × g for 5 min. Carefully decant the supernatant (= wash)
and save it for SDS-PAGE analysis.

6. Repeat steps 4 and 5 twice for a total of three washes.
7. Elute the bound protein by adding 0.5 ml of elution buffer per 1 ml slurry of Glutathione Sepharose
medium. Incubate at room temperature for 5 to 10 min, using gentle agitation such as end-over-end
rotation.
8. Sediment the medium by centrifugation at 500 × g for 5 min. Carefully decant the supernatant (= eluted
protein) and transfer to a clean tube.
9. Repeat steps 7 and 8 twice for a total of three elutions. Check the three eluates separately for purified
protein and pool those eluates containing protein.
Column purification of GST-tagged proteins using Glutathione Sepharose HP,

Glutathione Sepharose 4 FF, or Glutathione Sepharose 4B
Sample preparation
1. Prepare the cell lysate.
2. Centrifuge the cell lysate at high speed for 10 min at 4°C and pass it through a 0.45 µm filter before
applying to the Glutathione Sepharose column. If the sample is too viscous, to prevent it from clogging
the column dilute it with binding buffer, increase lysis treatment (sonication, homogenization), or add
Buffer preparation
Use high-purity water and chemicals, and pass all buffers through a 0.45 µm filter before use.
Column packing
Glutathione Sepharose media are supplied preswollen in 20% ethanol. Because of the nature of the
media, the steps for packing columns with them vary and are presented separately below.
Packing a column containing Glutathione Sepharose High Performance

Refer to Appendix 4 for general guidelines for column packing. Recommended lab-scale columns and
associated flow rates for Glutathione Sepharose High Performance are listed in Table 18.
Table 18. Recommended lab-scale columns for Glutathione Sepharose High Performance.
Empty column1 Packing flow rate (ml/min) Max. recommended flow rate
first step second step for purification (ml/min)
Tricorn 5/20 0.5 1 0.5
Tricorn 5/50 0.5 1 0.5
Tricorn 10/20 2 4 2
Tricorn 10/50 2 4 2
Tricorn 10/100 2 4 2
XK 16/20 5 9 5
XK 26/20 13 27 13
1 For inner diameter and maximum bed volumes and bed heights, refer to the catalog, BioDirectory, or Web site.

1. Equilibrate all materials to the temperature at which the purification will be performed.
2. Prepare a slurry by decanting an appropriate amount of the 20% ethanol solution and replacing it with
distilled water in a ratio of 75% settled medium to 25% distilled water.
water.
6. If using a packing reservoir, immediately fill the remainder of the column and reservoir with distilled
water. Mount the adapter or lid of the packing reservoir and connect the column to a pump. Avoid
trapping air bubbles under the adapter or in the inlet tubing.
Ideally, Sepharose High Performance media are packed in XK or Tricorn columns in a two-step
procedure: Do not exceed 1.0 bar (0.1 MPa) in the first step and 3.5 bar (0.35 MPa) in the second
step. If the packing equipment does not include a pressure gauge, use a packing flow rate of
5 ml/min (XK 16/20 column) or 2 ml/min (Tricorn 10/100 column) in the first step, and 9 ml/min
(XK 16/20 column) or 3.6 ml/min (Tricorn 10/100 column) in the second step. If the recommended
pressure or flow rate cannot be obtained, use the maximum flow rate your pump can deliver.
This should also give a well-packed bed.
For subsequent chromatography procedures, do not exceed 75% of the packing flow rate.
Packing a column containing Glutathione Sepharose 4 Fast Flow

Recommended columns:
• Tricorn 10/100 (10 mm i.d.) for bed volumes up to 8.5 ml at bed heights up to 10.8 cm.
• XK 16/20 (16 mm i.d.) for bed volumes up to 30 ml at bed heights up to 15 cm.
distilled water.

8. Maintain the packing flow for at least 3 bed volumes after a constant bed height is obtained. Mark the
Ideally, Fast Flow media are packed at constant pressure not exceeding 1 bar (0.1 MPa) in XK
columns. If the packing equipment does not include a pressure gauge, use a packing flow rate
of maximum 15 ml/min, 450 cm/h (XK 16/20 column) or 6 ml/min, 450 cm/h (Tricorn 10/100
column). If the recommended pressure or flow rate cannot be obtained, use the maximum flow
rate the pump can deliver. This should also give a reasonably well-packed bed.
Packing a column containing Glutathione Sepharose 4B

Recommended columns:
• Tricorn 10/100 (10 mm i.d.) for bed volumes up to 8.5 ml at bed heights up to 10.8 cm.
distilled water.
6. If using a packing reservoir, immediately fill the remainder of the column and reservoir with distilled
water. Mount the adapter or lid of the packing reservoir and connect the column to a pump. Avoid
trapping air bubbles under the adapter or in the inlet tubing.
8. Maintain the packing flow for at least 3 bed volumes after a constant bed height is obtained. Mark the
Ideally, 4B media are packed at constant pressure not exceeding 1 bar (0.1 MPa) in XK columns.
If the packing equipment does not include a pressure gauge, use a packing flow rate of maximum
2.5 ml/min, 75 cm/h (XK 16/20 column) or 1 ml/min, 75 cm/h (Tricorn 10/100 column). If the
recommended pressure or flow rate cannot be obtained, use the maximum flow rate the pump
can deliver. This should also give a reasonably well-packed bed.
Column purification
1. Equilibrate the column with approximately 5 column volumes of binding buffer.
3. Wash the column with 5 to 10 column volumes of binding buffer or until no material appears in the
flowthrough. Save the flowthrough for SDS-PAGE analysis to measure the binding efficiency to the
medium.
4. Elute the bound protein with 5 to 10 column volumes of elution buffer. Collect the fractions and check
separately for purified protein. Pool those fractions containing the GST-tagged target protein.

High-throughput screening using GST MultiTrap FF and
GST MultiTrap 4B 96-well filter plates
GST MultiTrap FF and GST MultiTrap 4B (Fig 53) are prepacked, disposable 96-well filter plates for
reproducible, high-throughput screening of GST-tagged proteins. Typical applications include expression
screening of different constructs, screening for solubility of proteins, and optimization of the conditions
for small-scale parallel purification. These filter plates simplify the purification screening and enrichment
of up to 0.5 mg of GST-tagged proteins/well. After thorough cell disruption, it is possible to apply up to
600 µl of unclarified lysate directly to the wells in the 96-well filter plate without precentrifugation and/or
filtration of the sample. It is recommended to extend the duration of mechanical/chemical lysis if the
sample is too viscous after lysis; alternatively, include nucleases to disrupt nucleic acids. The GST-tagged
proteins are eluted under mild, nondenaturing conditions that preserve protein function and antigenicity.
The plates are packed with the affinity media Glutathione Sepharose 4 Fast Flow (4% highly cross-
linked agarose beads) and Glutathione Sepharose 4B (4% agarose beads), respectively. Each well
contains 500 µl of a 10% slurry of Glutathione Sepharose 4 Fast Flow or Glutathione Sepharose 4B in
storage solution (50 µl of medium in 20% ethanol). Note that binding depends on flow and may vary
between proteins.
The 96-well filter plates with 800 µl wells are made of polypropylene and polyethylene. Characteristics
of GST MultiTrap FF and GST MultiTrap 4B are listed in Appendix 2.
Prepacked GST MultiTrap FF and GST MultiTrap 4B plates give high consistency in reproducibility well-
to-well and plate-to-plate. The repeatability of yield and purity of eluted protein is high. Automated
robotic systems as well as manual handling using centrifugation or vacuum pressure can be used.
The purification protocol can easily be scaled up because Glutathione Sepharose is available in larger
prepacked formats: GSTrap FF, and GSTrap 4B (1-ml and 5-ml columns) and GSTPrep FF 16/10
(20-ml column). See later in this chapter for a discussion of these products.
Fig 53. GST MultiTrap FF and GST MultiTrap 4B 96-well filter plates.

Sample preparation
Lysis with commercial kits could give large cell debris particles that may interfere with drainage
of the wells during purification. This problem can be solved by centrifugation or filtration of the
sample before adding it to the wells.
After thorough cell disruption, it is possible to apply unclarified lysate directly to the wells
without pre-centrifugation and/or filtration of the sample. Apply the unclarified lysate to the
wells directly after preparation, as the lysate may precipitate unless used immediately or frozen
before use. New lysing of the sample can then prevent clogging of the wells when loading
the plate.
If the sample is too viscous, an extension of the duration of mechanical treatment of the sample
to ensure a more complete lysis is recommended (keep the sample on ice to prevent overheating).
Buffer preparation
1 to 20 mM DTT can be included in the binding and elution buffers. However, this may result in
lower yield of GST-tagged protein.
Centrifugation procedure for high-throughput screening

Note: If the medium has dried out in one or several wells, add buffer to rehydrate it. The performance of
the medium is not affected.
5. Centrifuge the filter plate for 2 min at 500 × g to remove the ethanol storage solution from the medium.
6. Add 500 µl of deionized water to each well. Centrifuge the plate for 2 min at 500 × g.
7. Add 500 µl of binding buffer to each well to equilibrate the medium. Centrifuge for 2 min at 500 × g.
Repeat this step once. The filter plate is now ready for use.

Centrifugation procedure
Do not apply more than 700 × g during centrifugation.
for 3 min.
Note: If the yield of protein is too low, increase the incubation time and/or gently agitate the filter plate to
effect mixing.
2. Centrifuge the plate at 100 × g for 4 min or until all the wells are empty. Discard the flowthrough.
3. Add 500 µl of binding buffer per well to wash out any unbound sample. Centrifuge at 500 × g for 2 min.
Repeat once or until all unbound sample is removed.
Note: An A280 reading of < 0.1 of the collected liquid is required to obtain high purity. If necessary, change
the collection plate between each elution to prevent unnecessary dilution of the target protein.
Note: The volume of elution buffer can be varied (50 µl to 100 µl per well), depending on the concentration
of target protein required.
5. Change the collection plate and centrifuge at 500 × g for 2 min to collect the eluted protein. Repeat twice
or until all of the target protein has been eluted.
If the yield of eluted target protein is low, the incubation time can be increased.
Vacuum procedure for high-throughput screening

If problems with foaming, reproducibility, or bubbles in the collection plate occur using vacuum,
the centrifugation procedure should be considered. The distance between the filter plate and
the collection plate is critical; adjust the distance if necessary.

Note: If the medium has dried out in one or several wells, add buffer to rehydrate it. The performance of
the medium is not affected.
5. Set the vacuum to -0.15 bar. Place the filter plate and collection plate on the vacuum manifold to remove
the ethanol storage solution from the medium.
6. Add 500 µl of deionized water to each well. Apply a vacuum to remove the water from the wells.
7. Add 500 µl of binding buffer to each well to equilibrate the medium. Apply a vacuum as in step 5. Repeat
this step once. The filter plate is now ready for use.

Vacuum procedure
If a robotic system is used for purification, the vacuum must be adjusted according to methods
applicable to the system.
Do not apply a pressure in excess of -0.5 bar during vacuum operation.

for 3 min.
Note: If the yield of protein is too low, increase the incubation time and/or gently agitate the filter plate.
2. Apply a vacuum of -0.15 bar until all the wells are empty. Slowly increase the vacuum to -0.30 bar and
turn off the vacuum after approximately 5 sec. Discard the flowthrough.
Increasing the vacuum too quickly can result in foaming under the filter plate and subsequent
cross-contamination of samples.
3. Add 500 µl of binding buffer per well to wash out any unbound sample. Apply a vacuum of -0.15 bar as in
step 2. Repeat once or until all unbound sample is removed.
Note: An A280 reading of < 0.1 of the collected liquid is required to obtain high purity. If necessary, change
the collection plate between each elution to prevent unnecessary dilution of the target protein.
Note: The volume of elution buffer can be varied (50 µl to 100 µl per well), depending on the concentration
of target protein required.
5. Change the collection plate and apply a vacuum of -0.15 bar to collect the eluted protein. Repeat twice or
until all of the target protein has been eluted.
If the yield of eluted target protein is low, the incubation time can be increased.

Application example
High-throughput screening and purification of GST-hippocalcin using GST MultiTrap FF
GST MultiTrap FF and GST MultiTrap 4B allow reproducible, high-throughput screening and rapid
parallel purification of GST-tagged proteins, using robotic systems, centrifugation, or manual vacuum
manifolds. In this example, the conditions for binding buffer were optimized for purification of GST-
hippocalcin using GST MultiTrap FF. A buffer-screening study to determine optimal buffer conditions
for purification was designed based on the parameters of buffer, pH, sodium chloride, glycerol, DTT,
and glutathione. A comparison between sonication and use of a commercial cell lysis kit was also
performed. Factorial design (design-of-experiments) and statistical analysis were performed using
MODDE™ software (Umetrics). The different buffer conditions and sample preparation methods were
applied randomly on the filter plate.
The presence of glutathione in sample and binding buffer (also used as wash buffer) decreased yield
of purified GST-hippocalcin significantly, while the type of buffer used had no effect on yield. Low pH
improved yield whereas high pH (8.0) affected yield negatively. No significant effect on purity (Fig 54)
was seen with changing pH. Additives such as DTT, glycerol, and NaCl did not significantly affect yield
or purity of this particular protein.
The screening results showed that the optimal buffer conditions for purifying GST-hippocalcin with
highest yield and purity were: 10 to 20 mM sodium phosphate, 140 to 400 mM NaCl, pH 6.2 to 7.0 (data
not shown). Results reflecting sample preparation showed in this case that both the commercial cell
lysis kit and sonication can be used to lyse E. coli without significantly affecting the purification result.
96-well filter plate: GST MultiTrap FF

Sample: Unclarified E. coli BL21 lysate containing
GST-tagged hippocalcin, Mr 43 000
Sample preparation: Lysis using a commercial cell lysis kit and
sonication were compared. Both methods
were performed according to standard
protocols.
Sample volume: 500 µl
Elution volume: 3 × 200 µl
Binding buffer: Parameters varied and randomly tested:
10 to 20 mM PBS; 50 to100 mM Tris-HCl;
pH 6.2 to 8.0; 140 to 400 mM NaCl; 0 to
5 mM DTT; 0% to 5% glycerol and 0 to
2 mM glutathione
Elution buffer: 50 mM Tris-HCl, 10 mM reduced
glutathione, pH 8.0 Lanes
Elution method: Centrifugation 1. LMW markers
Purification protocol: According to GST MultiTrap instructions, 2. Start material
28-4070-75
3. Sonication, 10 mM PBS, 140 mM NaCl, pH 7.4
Data evaluation: MODDE software, UV-spectrometry (A280),
4. Cell lysis kit, 10 mM PBS, 140 mM NaCl, pH 7.4
SDS-PAGE
5. Cell lysis kit, 10 mM PBS, 400 mM NaCl, 2 mM glutathione,
5% glycerol, pH 8
Mr 6. Sonication, 20 mM PBS, 400 mM NaCl, 5% glycerol, pH 6.2
97 000 7. Sonication, 20 mM PBS, 400 mM NaCl, 2 mM glutathione,
66 000 pH 8
8. Sonication, 50 mM Tris-HCl, 400 mM NaCl, 5% glycerol,
45 000 pH 6.2
9. Sonication, 50 mM Tris-HCl, pH 8
30 000
10. Sonication, 50 mM Tris-HCl, 140 mM NaCl,
20 100 2 mM glutathione, 5 mM DTT, 5% glycerol, pH 8
14 400 11. Sonication, 100 mM Tris-HCl, 140 mM NaCl, 5 mM DTT,
pH 6.2
12. Sonication, 100 mM Tris-HCl, 270 mM NaCl,
1 2 3 4 5 6 7 8 9 10 11 12 1 mM glutathione, 2.5 mM DTT, 2.5% glycerol, pH 7.4
Fig 54. SDS-PAGE (reducing conditions, ExcelGel SDS Gradient 8–18; Coomassie staining) of collected fractions of eluted GST-hippocalcin
from some of the GST MultiTrap FF filter plate wells.

Minipreps using the GST SpinTrap Purification Module
The GST SpinTrap Purification Module is useful for screening small or large numbers of bacterial lysates
and for checking samples during the optimization of expression or purification conditions. Each module
contains reagents sufficient for 50 purifications using SpinTrap columns prepacked with Glutathione
Sepharose 4B. Sample application, washing, and elution can be performed using a standard
microcentrifuge.
Purification of multiple samples using GST SpinTrap columns

with a microcentrifuge
Each SpinTrap column contains 50 µl of Glutathione Sepharose 4B, sufficient for purifying up to
400 µg of recombinant GST.
Do not apply more than 600 µl of sample at a time to a GST SpinTrap column. This procedure
will accommodate lysates produced from 2 to 12 ml of culture.
Components in GST SpinTrap Purification Module

10× PBS: 1.4 M NaCl, 27 mM KCl, 100 mM Na 2HPO4, 18 mM KH2PO4, pH 7.3.
To prepare 1× PBS for use, dilute 10× PBS with water. Store at 4°C.
Reduced glutathione: 0.154 g. To prepare elution buffer, pour the entire 50 ml volume of dilution buffer
supplied with the module into the bottle containing the reduced glutathione. Shake until completely dissolved.
Store as 1 to 20 ml aliquots at -20°C.
Dilution buffer: 50 mM Tris-HCl, pH 8.0
SpinTrap columns: 50 units
Procedure
1. Resuspend the Glutathione Sepharose 4B in each column by vortexing gently.
2. Loosen the column caps one-fourth turn. Remove (and save) bottom closures.
3. Place each column into a clean 1.5- or 2-ml microcentrifuge tube. Spin for 1 min at 735 × g.
4. Discard the buffer from each centrifuge tube and replace the bottom closures. Remove the lids.
5. Apply up to 600 µl of lysate to a column.
6. Recap each column securely. Incubate at room temperature for 5 to 10 min while mixing gently by
repeated inversion.
7. Remove (and save) the top caps and bottom closures. Place each column into a clean, prelabeled 1.5- or
2-ml microcentrifuge tube.
8. Spin for 1 min at 735 × g to collect the flowthrough.
9. Place each column into a clean, prelabeled 1.5- or 2-ml microcentrifuge tube.
10. Apply 600 µl of 1× PBS wash buffer to each column and repeat the spin procedure. Additional 600 µl
washes with 1× PBS can be performed if desired.
11. Add 100 to 200 µl of elution buffer to each column. Replace top caps and bottom closures. Incubate at
room temperature for 5 to 10 min with gentle agitation.
12. Remove and discard top caps and bottom closures and place each column into a clean 1.5- or 2-ml
microcentrifuge tube.
13. Spin all columns again to collect the eluates. Save eluates for analysis.
Yields of tagged protein can be increased by repeating the elution step two or three times and
pooling the eluates.

Purification using GSTrap HP, GSTrap FF,
and GSTrap 4B columns
GSTrap affinity columns are specially designed 1-ml and 5-ml HiTrap columns packed with Glutathione
Sepharose HP, FF, or 4B media. Refer to the selection guide in Table 16 for a summary of their differences
and to Appendix 2 for a list of key characteristics of each.
Sample application, washing, and elution can be performed using a syringe with a supplied connector,
a peristaltic pump, or a liquid chromatography system such as ÄKTAdesign (see Table 8 for equipment
choices). For easy scale-up, two to three columns can be connected together in series simply by screwing
the end of one column into the top of the next.
Figure 55 shows a schematic representation of the simple steps needed for successful purification using
GSTrap columns.
Equilibrate column Apply sample Elute
with wash with with
binding buffer binding buffer elution buffer
3 min 5 to 15 min 2 min
Waste Collect flowthrough Collect fractions
Fig 55. GSTrap HP, GSTrap 4B, and GSTrap FF 1-ml and 5-ml columns allow convenient and simple one-step purification of GST-tagged
proteins. Simple purification of GST-tagged proteins is shown at right.
The GSTrap HP, FF, and 4B columns are made of polypropylene, which is biocompatible and non-
interactive with biomolecules. The top and bottom frits are manufactured from porous polyethylene.
Columns are delivered with a stopper on the inlet and a snap-off end on the outlet. Every package
includes all necessary components for connection of the columns to different types of equipment.
Note that GSTrap columns cannot be opened or refilled.
GSTrap columns are directly compatible with existing purification protocols for GST-tagged proteins,
including on-column proteolytic cleavage methods. If removal of the GST moiety is required, the tagged
protein can be digested with an appropriate site-specific protease while bound to the medium or,
alternatively, after elution (see later in this chapter). On-column cleavage eliminates the extra step of
separating the released protein from GST because the GST moiety remains bound. For quick scale-up
of purifications, two or three GSTrap columns (1 ml or 5 ml) can be connected in series (back pressure
will increase).
One of the three media, Glutathione Sepharose 4 Fast Flow, is also available in prepacked 20-ml
GSTPrep FF 16/10 columns (see page 134). All three are available in lab packs (varying from 25 to
500 ml) for packing in a column of the user's choice.
The media are very stable and the purification process very reproducible. This can be seen from the
results of an experiment in which E. coli homogenates containing GST-hippocalcin (Mr 43 000) were
repeatedly purified 10 times on the same column without cleaning between runs. The 10 overlaid
chromatograms (Fig 56A) show a near perfect match, indicating little or no variation in binding capacity
and stability of the medium. SDS-PAGE analysis (Fig 56B) also indicates no changes in purity or binding
capacity after 10 runs.

A)
mAU Column: GSTrap HP 1 ml
Sample: Clarified E. coli homogenate containing
expressed GST-hippocalcin, Mr 43 000
15000
Binding buffer: 10 mM sodium phosphate,
0.14 M NaCl, 1 mM DTT, pH 7.5
Elution buffer: 50 mM Tris-HCl, 10 mM reduced
10000
glutathione, 1 mM DTT, pH 8.0
Flow rate
5000 sample loading: 0.3 ml/min
wash and elution: 1 ml/min
Running temperature: Room temperature
0
0 5.0 10.0 15.0 ml
B)
Mr
97 000
66 000
GST-hippocalcin 45 000
30 000
20 100
14 400
1 2 3 4 5 6 7 8 9 10 11
Lanes 1 to 10. Eluted pooled fractions from runs 1 to 10, diluted 1:8.
Lane 11. LMW marker.
Fig 56. (A) Confirmation of the stability of Glutathione Sepharose High Performance prepacked in 1-ml GSTrap HP columns.
Chromatographic overlay of 10 repetitive purifications. (B) Coomassie-stained nonreduced SDS-PAGE (ExcelGel SDS Gradient 8–18) of
pooled fractions from repetitive purification runs shown in (A).
Sample preparation
The sample should be centrifuged and/or filtered through a 0.22-µm or a 0.45-µm filter immediately
before it is applied to the column. If the sample is too viscous, dilute it with binding buffer or
buffer exchange using HiTrap Desalting, PD-10 Desalting, or HiPrep 26/10 Desalting to prevent
clogging the column.
Buffer preparation
Elution buffer: 50 mM Tris-HCl, 10 to 20 mM reduced glutathione, pH 8.0

Purification
1. Fill the syringe or pump tubing with binding buffer. Remove the stopper and connect the column to the
syringe (use the connector supplied), laboratory pump, or chromatographic system “drop to drop” to
avoid introducing air into the column.
3. Equilibrate the column with 5 column volumes of binding buffer.
4. Apply the pretreated sample using a syringe fitted to the Luer connector or by pumping it onto the
column. For best results, use a flow rate of 0.2 to 1 ml/min (1-ml column) and 0.5 to 5 ml/min (5-ml
column) during sample application*.
5. Wash with 5 to 10 column volumes of binding buffer or until no material appears in the effluent. Maintain
a flow rate of 1 to 2 ml/min (1-ml column) and 5 to 10 ml/min (5-ml column) for washing.
Optional: Collect the flowthrough (in 1-ml fractions for the 1-ml column and 2-ml fractions for the 5-ml
column) and reserve until the procedure has been successfully completed. Retain a sample for analysis
by SDS-PAGE or by CDNB assay to measure the efficiency of protein binding to the medium.
6. Elute with 5 to 10 column volumes of elution buffer. Maintain a flow rate of 0.2 to 1 ml/min (1-ml column)
and 0.5 to 5 ml/min (5-ml column) for elution.
7. After elution, regenerate the column by washing it with 3 to 5 column volumes of binding buffer. The column
is now ready for a new purification.
A) B) C)
Fig 57. Using a GSTrap column with a syringe. A) Prepare buffers and sample. Remove the column’s top cap and snap off the end.
B) Load the sample and begin collecting fractions. C) Wash and elute and continue collecting fractions.
Volumes and times used for elution may vary among tagged proteins. Additional elutions with higher
concentrations of glutathione may be required. Flowthrough, wash, and eluted material from the
column should be monitored for GST-tagged proteins using SDS-PAGE in combination with Western
blotting, if necessary.
Flow rate will affect the binding and elution of GST-tagged proteins to the medium. Due to the
relatively slow binding kinetics between GST and glutathione, it is important to keep the flow
rate low for maximum binding capacity. Protein characteristics, pH, and temperature are other
factors that may affect the binding capacity. However, when working with sensitive proteins,
higher flow rates are recommended to minimize purification time. Combining two or three
columns in tandem would increase residence time for sample passing the column, thus allowing
higher flow rates to be used.
The reuse of GSTrap HP, FF, or 4B columns depends on the nature of the sample and should only
be performed with identical samples to prevent cross-contamination.

1. High-performance purification of GST-hippocalcin using 1-ml and 5-ml GSTrap HP columns
Glutathione Sepharose High Performance is easy to use for one-step purification of GST-tagged proteins.
The following data shows the results from experiments using both GSTrap HP 1 ml and 5 ml.
In this study, 5 ml and 25 ml of E. coli homogenate containing GST-hippocalcin was loaded on GSTrap
HP 1-ml and 5-ml columns, respectively. Figure 58A–B shows the chromatograms from the two runs.
The amount of protein in the eluted peaks was calculated as 6.5 mg and 39.7 mg, respectively.
The SDS-polyacrylamide gel shows GST-hippocalcin analyzed under nonreducing and reducing buffer
conditions (Fig 58C). Each well was loaded with 10 µg of protein. The SDS-polyacrylamide gel also
shows that free GST is expressed. The presence of reducing agent led to the removal of high-molecular-
weight bands, which may correspond to GST-tagged protein that associated by oxidation of the free
sulfhydryl groups.
A) mAU Columns: GSTrap HP 1 ml and GSTrap HP 5 ml
Sample: Clarified E. coli homogenate containing
expressed GST-hippocalcin, Mr 43 000
3500 Sample volumes: GSTrap HP 1 ml: 5 ml
3000 5 ml: 25 ml
6.5 mg Binding buffer: 10 mM sodium phosphate, 0.14 M NaCl,
2500 pH 7.4
2000 Elution buffer: 50 mM Tris-HCl, 10 mM reduced glutathione,
pH 8.0
1500 Flow rate
1000 sample loading: GSTrap HP 1 ml: 0.3 ml/min
5 ml: 1.6 ml/min
500 wash and elution: GSTrap HP 1 ml: 1 ml/min
0 5 ml: 4 ml/min
0.0 5.0 10.0 15.0 ml Running
temperature: Room temperature
B) mAU
3500
3000
39.7 mg
2500
2000
1500
1000
500
0
0.0 20.0 40.0 60.0 80.0 ml
C)
Mr
97 000
66 000
45 000 GST-hippocalcin
30 000
GST Lanes
2. Eluted pool from GSTrap HP 1 ml, nonreduced
14 400
3. “ 5 ml, nonreduced
4. “ 1 ml, reduced
1 2 3 4 5 5. “ 5 ml, reduced
Fig 58. Scale-up from (A) GSTrap HP 1 ml to (B) GSTrap HP 5 ml. (C) Coomassie-stained reduced and nonreduced SDS-PAGE (ExcelGel
SDS Gradient 8–18) of fractions from purification shown in Fig 58A–B.

2. Fast purification of a GST-tagged protein using GSTrap FF 1-ml and 5-ml columns
A GST-tagged protein was purified from 8 ml and 40 ml of a clarified cell lysate using GSTrap FF 1-ml
and 5-ml columns, respectively. Samples were applied to columns preequilibrated with PBS (pH 7.3).
After washing the columns with 10 column volumes of PBS, GST-tagged protein was eluted using
reduced glutathione (Fig 59). Each run was completed in 25 min using ÄKTAexplorer 10. Analysis by
SDS-PAGE indicated the isolation of highly pure GST-tagged protein (not shown). Yields of tagged
proteins were 2.7 mg from GSTrap FF 1-ml and 13.4 mg from GSTrap FF 5-ml.
A) B)
A 280 % A 280 %
Elution Elution Elution Elution
buffer buffer 3.5 buffer buffer
3.5
3.0
3.0 100
100 Wash
2.5
2.5 80
Wash 80 13.4 mg pure
2.7 mg pure 2.0
2.0 GST-tagged GST-tagged
60 protein 60
protein 1.5
1.5
40 40
1.0 1.0
20 0.5 20
0.5
0 0 0 0
5 10 15 20 ml 20 40 60 80 100 ml
5 10 15 20 min 4 8 12 16 20 min
Sample: 8 ml clarified E. coli lysate Sample: 40 ml clarified E. coli lysate

Column: GSTrap FF 1 ml Column: GSTrap FF 5 ml
Binding buffer: PBS (150 mM NaCl, Binding buffer: PBS (150 mM NaCl,
20 mM phosphate buffer, pH 7.3) 20 mM phosphate buffer, pH 7.3)
Elution buffer: 10 mM reduced glutathione, Elution buffer: 10 mM reduced glutathione,
50 mM Tris-HCl, pH 8.0 50 mM Tris-HCl, pH 8.0
Flow rate: 1 ml/min Flow rate: 5 ml/min
Chromatographic Chromatographic
procedure: 4 column volumes (CV) binding buffer, procedure: 4 column volumes (CV) binding buffer,
8 ml sample 40 ml sample
10 CV binding buffer, 5 CV elution buffer 10 CV binding buffer, 5 CV elution buffer
5 CV binding buffer 5 CV binding buffer
System: ÄKTAexplorer 10 System: ÄKTAexplorer 10
Fig 59. Purification of a GST-tagged protein on GSTrap FF 1-ml and 5-ml columns in combination with ÄKTAexplorer 10. Cytoplasmic
extract (8 and 40 ml) from E. coli expressing a GST-tagged protein were applied to GSTrap FF 1-ml (A) and GSTrap FF 5-ml (B),
respectively.

3. Two-step, automated purification using ÄKTAxpress
A two-step, automated purification of GST-hippocalcin from clarified E. coli lysate was performed on
ÄKTAxpress. A GSTrap 4B 1-ml column was used in the first affinity chromatography capture step and
a HiLoad 16/60 Superdex 200 pg column for the polishing step using gel filtration.
Reducing agent (DTT) was included in both sample and buffers. ÄKTAxpress enabled automated loading
of eluted fractions of the target protein from the capture step (GSTrap 4B) onto the gel filtration column.
Lysis of E. coli containing GST-hippocalcin was performed enzymatically followed by sonication. The
lysate was clarified by centrifugation and filtration, and 5 ml of the clarified lysate was loaded on the
1-ml GSTrap 4B column. Chromatograms from the automated two-step purification, as well as SDS-
PAGE of the eluted pool of target protein, are shown in Figure 60. Two peaks were obtained after gel
filtration: one small and one large. According to SDS-PAGE (only the pool of the large peak is shown,
Fig 60B), both peaks contained GST-hippocalcin. From evaluation of the gel filtration step, the large
peak seemed to be the dimer of GST-hippocalcin. The small peak is possibly a larger aggregate of
GST-hippocalcin. The purity of the GST-hippocalcin was good (Fig 60C).
Yield of eluted GST-hippocalcin, determined by absorbance at 280 nm using UNICORN software,
was 6.4 mg.
This application shows the benefit of using a two-step purification for increasing the purity of
GST-hippocalcin.
Columns: GSTrap 4B, 1 ml C)
HiLoad 16/60 Superdex 200 pg, 120 ml
Sample: Clarified E. coli lysate containing expressed
GST-hippocalcin, Mr 43 000 Mr
Sample volume: 5 ml (GSTrap 4B) 97 000
Binding buffer: 10 mM sodium phosphate, 140 mM NaCl, 20 mM DTT,
pH 7.4 66 000
Elution buffer: 50 mM Tris-HCl, 20 mM glutathione, 20 mM DTT,
pH 8.0
Buffer gel filtration: 10 mM sodium phosphate, 140 mM NaCl, 20 mM DTT, 45 000
pH 7.4
Flow rate: Sample loading, 0.3 ml/min (GSTrap 4B) 30 000
Wash and elution, 1 ml/min (GSTrap 4B)
1.5 ml/min (HiLoad 16/60 Superdex 200 pg)
Running temperature: 22°C 20 100
System: ÄKTAxpress
14 400
B) Dimers of GST-hippocalcin
mAU
Large aggregates of 1 2 3 4
250 GST-hippocalcin
200
Lanes
A)
150 1. LMW markers
mAU 100 2. Start material diluted 1:10
50 3. Flowthrough from affinity chromatography using
1500 0
130 150 170 ml
GSTrap 4B, diluted 1:3
4. GST-hippocalcin pool from gel filtration, undiluted.
1000
500
0
50 100 150 200 ml
AC Gel filtration
Fig 60. (A) Purification of GST-hippocalcin from E. coli lysate using an automated two-step purification on ÄKTAxpress. (B) Enlargement
of the peak from the gel filtration step revealed large aggregates and dimers of purified GST-hippocalcin. (C) SDS-PAGE (ExcelGel SDS
Gradient 8–18%) showing final purity of GST-hippocalcin (lane 4).

Purification of a GST-tagged protein using GSTrap FF 1 ml with
ÄKTAprime plus
This procedure uses a GSTrap FF 1-ml column but also can be used with GSTrap HP or GSTrap
4B 1-ml columns.
Sample preparation
The sample should be centrifuged and/or filtered through a 0.45-µm filter immediately before it
is applied to the column. If the sample is too viscous, dilute it with binding buffer or buffer
exchange using HiTrap Desalting, PD-10 Desalting, or HiPrep 26/10 Desalting to prevent
Buffer preparation
Binding buffer (port A1): 20 mM sodium phosphate, 0.15 M NaCl, pH 7.3

Elution buffer (port B): 50 mM Tris-HCl, 10 mM reduced glutathione, pH 8.0
System preparation
Once the system is prepared, the remaining steps (under Selecting Application Template and
starting the method) will be performed automatically.
1. Place each inlet tubing from port A (8-port valve) in the binding buffer and the tubing from port B (2-port
valve) in the elution buffer.
4. Fill the fraction collector rack with 18-mm tubes (minimum 10) and position the white plate on the
fractionation arm against the first tube.
5. Connect a sample loop large enough for your sample between port 2 and 6 on the injection valve. Use a
Note: If a Superloop is needed, additional information is supplied in the instructions for Superloop.

Selecting Application Template and starting the method
2. Use the arrow and OK buttons to move in the menu tree until you find GST-tag Purification GSTrap.
Set Sample Inj. Vol
Templates
(00.0 ml) 00.0

Press OK to start
GST-tag Purification
Run data displayed
GSTrap
A)
%B Elution
100
Priming &
equilibration
50
Sample
Reequilibration
Wash
11 10 11 6 Min
B)
AU280 UV 280 nm
Programmed %B %B Sample: Clarified homogenate of E. coli
expressing GST-tagged protein
1.6 Column: GSTrap FF 1 ml
80 Binding buffer (port A1): 20 mM sodium phosphate,
0.15 M NaCl, pH 7.3
Elution buffer (port B): 50 mM Tris-HCl, 10 mM reduced
60 glutathione, pH 8.0
0.8 GST-
tagged
protein 40
Inject 20
0 0
0 12.5 25 min
Fig 61. (A) Theoretical gradient in GST-tag Purification GSTrap template. Total separation time = 37 min + sample application time.
(B) Typical results for purification of a GST-tagged protein.

Preparative purification using GSTPrep FF 16/10 column
GSTPrep FF 16/10 columns are based on the 20-ml HiPrep column design, ready to use for easy, one-
step preparative purification of GST-tagged proteins, other glutathione S-transferases, and glutathione
binding proteins. Prepacked with Glutathione Sepharose 4 Fast Flow, the columns exhibit high binding
capacity and excellent flow properties. For easy scale-up, columns can be connected in series.
Fig 62. GSTPrep FF 16/10 column.
Separations can be easily achieved using a chromatography system such as ÄKTAdesign. Refer to
Table 8 for a selection guide to purification equipment and to Appendix 2 for a list of GSTPrep FF
16/10 column parameters.
Glutathione Sepharose 4 Fast Flow is also available as prepacked 1-ml and 5-ml GSTrap FF columns,
as prepacked 96-well filter plates, GST MultiTrap FF, and as a bulk medium in lab packs (25, 100, and
500 ml) for packing columns or batch purifications. Note that GSTPrep FF 16/10 columns cannot be
opened or refilled.
Sample preparation
The sample should be centrifuged and/or filtered through a 0.22-µm or a 0.45-µm filter immediately
before it is applied to the column. If the sample is too viscous, dilute it with binding buffer or
buffer exchange using HiTrap Desalting, PD-10 Desalting, or HiPrep 26/10 Desalting to prevent
Buffer preparation

Purification
1. Apply the centrifuged and/or filtered sample (in binding buffer) to the column at a flow rate of
1 to 5 ml/min (30 to 150 cm/h).
2. Wash the column with 100 to 200 ml of binding buffer at 2 to 10 ml/min (60 to 300 cm/h).
3. Elute the bound protein with 100 to 200 ml of elution buffer at a flow rate of 2 to 10 ml/min
(60 to 300 cm/h).
4. Equilibrate the column with 60 to 100 ml of binding buffer at a flow rate of 2 to 10 ml/min (60 to 300 cm/h).
The column is now ready for a new purification.
Due to the relatively slow binding kinetics between GST and glutathione, it is important to keep
the flow rate low during sample loading/elution. The binding capacity may be different for
different proteins. The yield may therefore vary between proteins if sample load is close to the
capacity of the column.
Optional: Collect the flowthrough and reserve until the procedure has been successfully
completed. Retain a sample for analysis by SDS-PAGE or by CDNB assay to measure the
efficiency of protein binding to the medium
Reuse of any purification column depends on the nature of the sample and should only be
performed with identical tagged proteins to prevent cross-contamination.
Application example
1. Purification and scale-up of two GST-tagged proteins using 1-ml and 5-ml GSTrap FF
columns and GSTPrep FF 16/10 column
Glutathione Sepharose 4 Fast Flow is easy to use for one-step purification of GST-tagged proteins.
Figures 63A-C and 64A-C show scale-up studies on GSTrap FF 1 ml, GSTrap FF 5 ml, and GSTPrep FF
16/10. Two different GST tagged proteins were purified: GST-DemA and GST-Purα. The gene encoding
for DemA was isolated from Streptococcus dysgalactiae. DemA is a fibrinogen-binding protein that
shows both plasma protein binding properties and sequence similarities with the M and M-like proteins
of other streptococcal species. Purα has been shown to be involved in transcriptional regulation.
E. coli expressing the GST-tagged proteins was resuspended (1 g/5 ml) in PBS (140 mM NaCl, 2.7 mM KCl,
10 mM Na2HPO4, 1.8 mM KH2PO4, pH 7.4) supplemented with 1 mM PMSF, 1 mM DTT, 100 mM MgCl2,
1 U/ml RNase A and 13 U/ml DNase I. The cells were lysed by sonication with a Vibracell™ ultrasonic
processor for 3 min, amplitude 50%. The cell extract was kept on ice during the sonication. Cell debris
was removed by centrifugation at 48 000 x g, 4°C for 30 min. The supernatant was applied to the
column after passage through a 0.45 µm filter.
The following purification procedures were performed using ÄKTAexplorer 100 chromatography system.
The columns, GSTrap FF 1 ml, GSTrap FF 5 ml, and GSTPrep FF 16/10 were equilibrated with 5 column
volumes (CV) of PBS, pH 7.4 and the prepared sample was applied to the columns. The columns were
washed with 10 CV of PBS (GST-DemA) and 20 CV of (GST-Purα) and eluted using 7 CV of Tris-HCl,
pH 8.0 including 10 mM reduced glutathione. The purity of eluted proteins was analyzed by SDS-PAGE
(see Figs 63D and 64D).
The main parameter in this scale-up study was the residence time (i.e., the period of time the sample is
in contact with the medium). The residence time was the same for the GSTrap FF 1-ml and 5-ml columns
whereas it was twice as long for the GSTPrep FF 16/10 column (20-ml column volume) compared with
GSTrap FF 5-ml columns due to the difference in column length vs. column diameter. The amount of

protein bound differed between GST-DemA and GST-Purα, due to protein dependent binding charac-
teristics. Some of the applied protein was found in the flowthrough as an effect of the low turnover rate
number of GST. The amount of eluted GST-tagged proteins increased proportionally with increased
column volume and sample load.
A) A 280 Elution buffer Elution buffer A) GSTrap FF 1 ml

%B Column: GSTrap FF 1 ml
mAU 5.5 mg 100 Sample: 10 ml extract from E. coli expressing
Wash
1200 GST-DemA GST-DemA
80 Binding buffer: PBS (pH 7.4)
1000 Elution buffer: 50 mM Tris-HCl, pH 8.0 with 10 mM
800 60 reduced glutathione
Flow rate: Sample loading: 0.5 ml/min
600 40 Washing and elution: 1 ml/min
400 Chromatographic
20 procedure: 5 CV (CV= Column Volume) binding buffer,
200 10 ml sample, 10 CV binding buffer,
7 CV elution buffer, 5 CV binding buffer
0 0
0.0 10.0 20.0 30.0 ml System: ÄKTAexplorer 100
B) A 280 Elution buffer Elution buffer B) GSTrap FF 5 ml
mAU %B Column: GSTrap FF 5 ml
1400 100 Sample: 50 ml extract from E. coli expressing
20.9 mg GST-DemA
1200 GST-DemA
80 Binding buffer: PBS (pH 7.4)
1000 Wash Elution buffer: 50 mM Tris-HCl, pH 8.0 with
10 mM reduced glutathione
800 60 Flow rate: Sample loading: 2.5 ml/min
Washing and elution: 5 ml/min
600 40 Chromatographic
procedure: 5 CV binding buffer, 50 ml sample,
400 10 CV binding buffer, 7 CV elution buffer,
20 5 CV binding buffer
200
0 0
0 50 100 150 ml
C) A 280 C) GSTPrep FF 16/10
Elution buffer Elution buffer
mAU %B Column: GSTPrep FF 16/10
1400 Sample: 200 ml extract from E. coli expressing
100
111.0 mg GST-DemA
1200 GST-DemA Binding buffer: PBS (pH 7.4)
80 Elution buffer: 50 mM Tris-HCl, pH 8.0 with
1000 Wash
800 60 Flow rate: Sample loading: 5 ml/min
600 Chromatographic
40 procedure: 5 CV binding buffer, 200 ml sample,
200 System: ÄKTAexplorer 100
0 0
0 100 200 300 400 500 600 ml
D)
Mr
Lanes
97 000
1. LMW markers, reduced
66 000 2. Extract of E. coli expressing GST-DemA, 1 g cell paste/5 ml
GST-DemA
45 000 3. Flowthrough from GSTrap FF 1 ml
30 000 4. GST-DemA eluted from GSTrap FF 1 ml
5. Extract of E. coli expressing GST-DemA, 1 g cell paste/5 ml
14 400 7. GST-DemA eluted from GSTrap FF 5 ml
8. Extract of E. coli expressing GST-DemA, 1 g cell paste/5 ml
9. Flowthrough from GSTPrep FF 16/10
1 2 3 4 5 6 7 8 9 10 10. GST-DemA eluted from GSTPrep FF 16/10
Fig 63. Purification and scale-up of GST-DemA on (A) GSTrap FF 1 ml, (B) GSTrap FF 5 ml, and (C) GSTPrep FF 16/10. (D) SDS-PAGE analysis of
GST-DemA on ExcelGel Homogeneous 12.5% using Multiphor™ II followed by Coomassie staining. Due to the relatively slow kinetics of low
turnover rate number of GST and rather high load, some of the applied protein was found in the flowthrough.

A) A Elution buffer Elution buffer A) GSTrap FF 1 ml
280
mAU %B Column: GSTrap FF 1 ml
13.9 mg 100 Sample: 5 ml extract from E. coli expressing GST-Purα
GST-Purα Binding buffer: PBS (pH 7.4)
1500 80
Wash Elution buffer: 50 mM Tris-HCl, pH 8.0 with
60 Flow rate: Sample loading: 0.5 ml/min
1000
40 Chromatographic
5 CV binding buffer
0.0 10.0 20.0 30.0 ml
B) A 280 Elution buffer Elution buffer B) GSTrap FF 5 ml

%B Column: GSTrap FF 5 ml
mAU 100 Sample: 25 ml extract from E. coli expressing GST-Purα
Binding buffer: PBS (pH 7.4)
72.8 mg
1500 GST-Purα 80 Elution buffer: 50 mM Tris-HCl, pH 8.0 with
Wash Flow rate: Sample loading: 2.5 ml/min
60 Washing and elution: 5 ml/min
1000
Chromatographic
20 CV binding buffer, 7 CV elution buffer,
20 System: ÄKTAexplorer 100
0 0
0 50 100 150 ml
C) A 280 Elution buffer Elution buffer C) GSTPrep FF 16/10 (20-ml column volume)
%B Column: GSTPrep FF 16/10
mAU
100 Sample: 100 ml extract from E. coli expressing
2000 GST-Purα
346.4 mg
GST-Purα Binding buffer: PBS (pH 7.4)
80
Elution buffer: 50 mM Tris-HCl, pH 8.0 with
1500
Wash 10 mM reduced glutathione
60 Flow rate: Sample loading: 5 ml/min
1000
40 Chromatographic
procedure: 5 CV binding buffer, 100 ml sample,
20 CV binding buffer, 7 CV elution buffer,
500 20 5 CV binding buffer
0 0
0 100 200 300 400 500 600 ml
D)
Mr
97 000 Lanes
66 000 GST-Purα 1. LMW markers, reduced
2. Extract of E. coli expressing GST-Purα, 1 g cell paste/5 ml
30 000 4. GST-Purα eluted from GSTrap FF 1 ml
5. Extract of E. coli expressing GST-Purα, 1 g cell paste/5 ml
7. GST-Purα eluted from GSTrap FF 5 ml
14 400 8. Extract of E. coli expressing GST-Purα, 1 g cell paste/5 ml
9. Flowthrough from GSTPrep FF 16/10
1 2 3 4 5 6 7 8 9 10 10. GST-Purα eluted from GSTPrep FF 16/10
Fig 64. Purification and scale-up of GST-Purα on (A) GSTrap FF 1 ml, (B) GSTrap FF 5 ml, and (C) GSTPrep FF 16/10. (D) SDS-PAGE analysis
of GST-Purα on ExcelGel Homogeneous 12.5% using Multiphor II followed by Coomassie staining. Due to the low turnover rate number
of GST, some of the applied protein was found in the flowthrough.

Troubleshooting of purification methods
The troubleshooting guide below addresses problems common to the majority of purification methods
as well as problems specific to a particular method. In the latter case, the relevant method is indicated.

GST-tagged protein GST-tagged protein denatured by Use mild mechanical/chemical lysis
does not bind or binds mechanical lysis (e.g., sonication). conditions during cell lysis. Conditions for
very poorly. Too extensive lysis can denature lysis must be empirically determined.
the tagged protein and prevent it
from binding.
GST-tagged proteins have Add DTT to the sample prior to cell lysis and
aggregated in the sample, also add DTT to the buffers.
causing precipitation. Adding DTT to a final concentration of
1 to 20 mM may significantly increase
binding of some GST-tagged proteins.
Concentration of tagged protein Concentrate the sample. The binding
is too low. capacity is concentration dependent.
Low expressed proteins may not bind as
efficiently as highly expressed proteins,
therefore concentrating the sample may
improve binding.
The tagged protein may have Test the binding of GST from parental pGEX:
altered the conformation of GST, Prepare a sonicate of cells harboring the
thereby reducing the affinity for parental pGEX plasmid and check binding to
the GST-tagged protein. the medium. If GST produced from the
parental plasmid binds with high affinity,
the tagged protein may have altered the
conformation of GST, thereby reducing the
affinity for the GST-tagged protein.
Adequate results may be obtained by
reducing the temperature used for binding
to 4°C, and by limiting washing.
Equilibration time is too short. Ensure that the column has been
equilibrated with at least 5 column volumes
of a buffer pH 6.5 to 8.0 (e.g., PBS).
Binding of GST-tagged proteins Equilibrate with a buffer pH 6.5 to 8.0
is not efficient at pH less than 6.5 (e.g., PBS) before the clarified cell lysate
or greater than 8. is applied.
GSTrap column: Column needs Clean the column according to the standard
cleaning. cleaning protocol (see Appendix 2). If the
GSTrap column has already been used
several times, it may be necessary to use a
new one.
Glutathione Sepharose medium Use fresh Glutathione Sepharose medium.
has been used too many times. See also cleaning procedures in Appendix 2.
The flow rate used during sample Decrease the flow rate during sample
loading is too high. loading. One of the most important
parameters affecting the binding of GST-
tagged proteins to Glutathione Sepharose
is the flow rate. Due to the relatively slow
binding kinetics between GST and
glutathione, it is important to keep the flow
rate low during sample loading for maximum
binding capacity.

GSTrap columns on ÄKTAprime plus: Clogged column: Clean the column
The column or system is clogged, according to instructions. Make sure the
leading to high back pressure and sample has been centrifuged and/or filtered
no binding. through a 0.45 µm filter.
Clogged system: Replace the column
with a piece of tubing. Check pressure.
If back pressure > 0.3 MPa, clean system
according to manual.
GSTrap columns on ÄKTAprime plus: Check that the correct column is used.
The sample does not bind. Check that the inlet tubing from each
buffer is connected to the correct inlet port.
Check that the composition and pH of the
buffers are correct.
Check that the sample has been adjusted
to binding buffer conditions.
GST-tagged protein is The volume of elution buffer Increase the volume of elution buffer used.
not eluted efficiently. is insufficient. In some cases, especially after on-column
cleavage of a tagged protein, a larger
volume of buffer may be necessary to elute
the tagged protein.
The time allowed for elution Increase the time used for elution by
is insufficient. decreasing the flow rate during elution.
With GSTrap columns, for best results use
a flow rate of 0.2 to 1 ml/min (1-ml HiTrap
column) and 0.5 to 5 ml/min (5-ml HiTrap
column) during sample application. For
centrifugation methods, decrease the
centrifugation speed during elution.
The concentration of glutathione Increase the concentration of glutathione in
is insufficient. the elution buffer: The 10 mM recommended
in this protocol should be sufficient for most
applications, but exceptions exist. Try
50 mM Tris-HCl, 20 to 40 mM reduced
glutathione, pH 8.0 as elution buffer.
The pH of the elution buffer is Increase the pH of the elution buffer:
too low. Increasing the pH of the elution buffer to
pH 8 to 9 may improve elution without
requiring an increase in the concentration of
glutathione used for elution.
The ionic strength of the elution Increase the ionic strength of the elution
buffer is too low. buffer: Adding 0.1 to 0.2 M NaCl to the
elution buffer may also improve results.
The glutathione in the elution buffer Use fresh elution buffer.
is oxidized. Add DTT.
Nonspecific hydrophobic Add a nonionic detergent to the elution
interactions cause nonspecific buffer: Adding 0.1% Triton X-100 or
interaction with the medium 2% n-octylglucoside can significantly
or aggregation of proteins, improve elution of some GST-tagged
preventing solubilization and proteins.
elution of tagged proteins.
GSTrap columns on ÄKTAprime plus: Check that the inlet tubing from each
Poor elution. buffer is connected to the correct inlet port.
Check that the composition and pH of the
buffers are correct.
Use alternative elution conditions according
to the column instructions.

Multiple bands are Mr 70 000 protein copurifies with The Mr 70 000 protein is probably a protein
observed after the GST-tagged protein. product of the E. coli gene dnaK. This protein
electrophoresis/Western is involved in protein folding in E. coli. It has
blotting analysis of eluted been reported that this association can be
target protein. disrupted by incubating the tagged protein
in 50 mM Tris-HCl, 2 mM ATP, 10 mM MgSO4,
pH 7.4 for 10 min. at 37°C prior to loading.
Alternatively, remove the DnaK protein by
passing the tagged protein solution through
ATP-agarose or a similar purification
medium, or perform ion exchange.
Partial degradation of tagged Add a protease inhibitor: Multiple bands

proteins by proteases. may be a result of partial degradation of
tagged proteins by proteases. Adding
1 mM PMSF to the lysis solution may
improve results. A nontoxic, water-soluble
alternative to PMSF is 4-(2-aminoethyl)-
benzenesulfonyl fluoride hydrochloride
(AEBSF), commercially available as
Pefabloc SC from Roche Biochemicals.
Note: Serine protease inhibitors must be
removed prior to cleavage by thrombin or
Factor Xa. PreScission Protease is not a
consensus serine protease and is insensitive
to many of the protease inhibitors tested at
GE Healthcare.
PMSF is toxic, with acute effects.
Use Pefabloc whenever possible.
Proteolysis in the host bacteria. Use a protease-deficient host: Multiple
bands may be the result of proteolysis in
the host bacteria. If this is the case, the use
of a host-deficient strain may be required
(e.g., lon- or ompT). E. coli BL21 is provided
with the pGEX vectors. This strain is defective
in OmpT and Lon protease production.
Cell disruption during Decrease lysis time: Cell disruption is
mechanical lysis. apparent by partial clearing of the
suspension and can be checked by
microscopic examination. Adding lysozyme
(0.1 volume of a 10 mg/ml lysozyme
solution in 25 mM Tris-HCl, pH 8.0) prior to
mechanical lysis may improve results.
Avoid frothing as this may denature the
tagged protein. Over-lysis can also lead to
the copurification of host proteins with the
GST-tagged protein.

Chaperonins may have copurified. Include an additional purification step:
Additional bands may be caused by the
copurification of a variety of proteins known
as chaperonins, which are involved in the
correct folding of nascent proteins in E. coli.
These include, but may not be limited to:
DnaK (Mr ~ 70 000), DnaJ (Mr ~ 37 000), GrpE
(Mr ~ 40 000), GroEL (Mr ~ 57 000), and GroES
(Mr ~10 000). Several methods for purifying
GST-tagged proteins from these copurifying
proteins have been described.
Antibodies that react with various Cross-adsorb antibody with E. coli proteins:
E. coli proteins may be present in Depending on the source of the anti-GST
the tagged protein sample. antibody, it may contain antibodies that
react with various E. coli proteins that may
be present in the tagged protein sample.
Cross-adsorb the antibody with an E. coli
sonicate to remove anti-E. coli antibodies
from the preparation. Anti-GST antibody
from GE Healthcare has been cross-
adsorbed against E. coli proteins and tested
for its lack of nonspecific background
binding in Western blots.
Multiple bands are Proteolysis has occurred in the Determine when the bands appear: Test to
observed after host bacteria. be certain that additional bands are not
electrophoretic present prior to PreScission Protease,
analysis of cleaved thrombin, or Factor Xa cleavage. Such bands
target protein. may be the result of proteolysis in the host
bacteria.
Tagged partner may contain recognition
sequences for PreScission Protease,
thrombin, or Factor Xa: Check the
sequences. See the GST Gene Fusion
System Handbook for details.

Detection of GST-tagged proteins
Several methods are available for detection of GST-tagged proteins, with method selection largely
depending on the experimental situation. For example, SDS-PAGE analysis, although frequently used
for monitoring results during expression and purification may not be the method of choice for routine
monitoring of samples from high-throughput screening. Functional assays based on the properties of
the protein of interest (and not the GST tag) are useful, but must be developed for each specific protein.
These latter assays are not covered in this handbook.
Much of the information presented below can also be applied to detection of histidine-tagged proteins,
although the specific reagents will change.
GST 96-Well Detection Module for ELISA

The GST 96-Well Detection Module provides a highly sensitive enzyme-linked immunosorbent assay
(ELISA) for testing clarified lysates and intermediate purification fractions for the presence of GST-tagged
proteins (see Figs 65 and 66). Samples are applied directly into the wells of the plates, and GST-tagged
proteins are captured by specific binding to anti-GST antibody that is immobilized on the walls of each
well. After removal of unbound material by washing, the captured GST-tagged proteins are then
detected with HRP/Anti-GST conjugate provided in the module. Standard curves for quantitation of
tagged proteins can be constructed using purified recombinant GST, which is included as a control.
Each detection module contains reagents sufficient for 96 detections. Each plate is an array of 12 strips
with eight wells per strip, such that as few as eight samples (one strip) can be assayed at a time.
The GST 96-Well Detection Module can also be used with antibody directed against a GST fusion
partner to screen and identify clones expressing the desired GST-tagged protein.
A 450
1.50
0.75
0
0.01 0.1 1 10 100 1000 10 000
ng rGST/well
Fig 65. Sensitive detection of recombinant GST using the GST 96-Well Detection Module. Recombinant GST protein was prepared in
1× blocking buffer, and 100 µl volumes were applied directly to the wells of a GST 96-well capture plate. After 1 h binding at room
temperature, the wells were washed in wash buffer and incubated with a 1:1000 dilution of HRP/Anti-GST conjugate for 1 h. Detection
was performed using 3, 3',5,5'-tetramethyl benzidine (TMB) substrate, and the absorbance of each well was measured at 450 nm.

Fig 66. Screening of bacterial lysates for GST-tagged protein expression using the GST 96-Well Detection Module.
Each tagged protein is captured uniquely; therefore, if quantitation is required, prepare standards
of recombinant GST protein and the target tagged protein (if available) using a dilution series
from 1 ng/µl to 10 pg/µl in 1× blocking buffer. Include recombinant GST protein as a standard
control in every assay.
Prepare fresh buffers daily.
Components of GST 96-Well Detection Module

GST 96-Well Detection Plates (each well is coated with goat polyclonal anti-GST antibody, blocked, and dried)
Horseradish peroxidase conjugated to goat polyclonal anti-GST antibody (HRP/Anti-GST)
Purified recombinant GST standard protein
Additional reagents required for ELISA

PBS: 140 mM NaCl, 2.7 mM KCl, 10 mM Na2HPO4, 1.8 mM KH2PO4, pH 7.3
Wash buffer: 0.05% Tween 20 in PBS (500 ml/96-well plate). Store at room temperature until needed.
Blocking buffer (1×): 3% nonfat dry milk in PBS with 0.05% Tween 20 (10 ml/96-well plate)
Blocking buffer (2×): 6% nonfat dry milk in PBS with 0.1% Tween 20 (5 ml/96-well plate)
Substrate
Procedure
1. Bring each test sample to a final volume of 50 µl with 1× PBS.
2. Add 50 µl of 2× blocking buffer to each sample.
3. For screening, dilute the recombinant GST protein standard to 1 ng/100 µl in 1× blocking buffer.
4. For quantitation, prepare a dilution series from 1 ng/µl to 10 pg/µl in 1× blocking buffer for both the
recombinant GST protein and the target tagged protein (when available).
5. Remove one 96-well plate from its foil pouch.
If using fewer than 96 wells, carefully remove the well strips from the holder by pushing up on
the wells from below. Store unused well strips in the pouch with the desiccant provided.
6. Pipette 100 µl of sample into each well.
7. Incubate for 1 h at room temperature in a humidified container or incubator.
8. Invert the plate and flick sharply to empty the contents of the wells.

Biohazardous material should be pipetted or aspirated into a suitable container.
9. Blot the inverted plate or well strips onto a paper towel to remove excess liquid.
10. Wash each well five times with wash buffer by inverting and flicking out the contents each time.
11. Blot the inverted plate or well strips onto a paper towel to remove excess wash buffer.
12. Dilute the HRP/anti-GST conjugate 1:10 000 (1 µl:10 ml) in 1× blocking buffer.
One 96-well plate will require 10 ml of the diluted conjugate.

13. Add 100 µl of diluted HRP/anti-GST conjugate to each well and incubate for 1 h at room temperature in a
humidified container or incubator.
14. Empty the well contents and wash twice with wash buffer as previously described.
15. Add soluble horseradish peroxidase substrate* to each well and incubate according to the supplier’s
instructions.
*3,3',5,5'-tetramethyl benzidine (A450) and 2',2'-azino-bis (3-ethylbenzthiazoline-6-sulfonic acid)
diammonium salt (ABTS™) (A410) have been used successfully.
16. Read plate absorbance in a microplate reader or spectrophotometer.
GST Detection Module with CDNB enzymatic assay

GST-tagged proteins produced using pGEX vectors can be detected enzymatically using the GST
substrate 1-chloro-2,4 dinitrobenzene (CDNB), included in the GST Detection Module. The GST-mediated
reaction of CDNB with glutathione produces a conjugate that is measured by absorbance at 340 nm
using either a plate reader or a UV/vis spectrophotometer. Assay results are available in less than 10 min
for crude bacterial sonicates, column eluates, or purified GST-tagged protein. Figure 67 shows typical
results from a CDNB assay. Each GST Detection Module contains reagents sufficient for 50 assays.
A 340
0.6
Eluate (0.8 µg)
0.4
0.2
Sonicate (53 µg)
1 2 3 4
Time (min)
Fig 67. Typical results of a CDNB assay for GST-tagged proteins. 53 µg of total protein from an E. coli TG1/pGEX-4T-Luc sonicate and 0.8 µg
of total protein eluted from Glutathione Sepharose were assayed according to instructions included with the GST Detection Module.
Components of GST Detection Module used with the CDNB enzymatic assay
10× reaction buffer: 1 M KH2PO4 buffer, pH 6.5
CDNB: 100 mM 1-chloro-2,4-dinitrobenzene (CDNB) in ethanol
Reduced glutathione powder: Prepare a 100 mM solution by dissolving the glutathione in sterile distilled
water. Aliquot into microcentrifuge tubes. Store at -20°C. Avoid more than five freeze/thaw cycles.
CDNB is toxic. Avoid contact with eyes, skin and clothing. In case of accidental contact, flush
affected area with water. In case of ingestion, seek immediate medical attention.
pGEX-bearing cells must be lysed prior to performing a CDNB assay.

Steps
1. In a microcentrifuge tube, combine the following:
Distilled water 880 µl
10× reaction buffer 100 µl
CDNB 10 µl
Glutathione solution 10 µl
Total volume 1000 µl
2. Cap the tube and mix the contents by inverting several times.
CDNB may cause the solution to become slightly cloudy. However, the solution should clear
upon mixing.
3. Transfer 500 µl volumes of the above CDNB solution into two UV-transparent cuvettes labeled sample
and blank. Add sample (5 to 50 µl) to the sample cuvette. To the blank cuvette, add 1× reaction buffer
equal in volume to that of the sample in the sample cuvette.
4. Cover each cuvette with wax film and invert to mix.
5. Place the blank cuvette into the spectrophotometer and blank at 340 nm. Measure the absorbance of the
sample cuvette at 340 nm and simultaneously start a stopwatch or other timer.
6. Record absorbance readings at 340 nm at 1 min intervals for 5 min by first blanking the spectrophotometer
with the blank cuvette and then measuring the absorbance of the sample cuvette.
For analyses using 96-well plates, add samples to the cells first and add reagents second. Mix
the content of the wells using the pipette. Start measuring the absorbance in the plate reader.
7. Calculate the A340/min/ml sample as follows:
Calculations
A340(t2) – A340(t1)
∆A340/min/ml =
(t2 – t1)(ml sample added)
Where: A340 (t2) = absorbance at 340 nm at time t2 in min

A340 (t1) = absorbance at 340 nm at time t1 in min
∆A340/min/ml values can be used as a relative comparison of GST-tagged protein content

between samples of a given tagged protein.
Adapt the assay to give absolute concentrations of tagged proteins by constructing a standard
curve of ∆A340/min versus amount of tagged protein. Purified sample of the tagged protein is
required to construct the curve.
The assay detects active GST. Additional GST-tagged protein may be present that is not active.
Activity of the GST moiety can be affected by folding of the fusion partner. Absorbance readings
obtained for a given tagged protein may not reflect the actual amount of tagged protein present.

Western blot
Expression and purification of GST-tagged proteins can be monitored by Western blot analysis, using
ECL, ECL Plus, or ECL Advance detection systems to enhance sensitivity. The combination of Western
blot detection and total protein staining of the SDS-PAGE (Coomassie or silver staining) gives a powerful
control of purification results.
Reagents required
Anti-GST antibody (goat polyclonal)
Blocking/incubation buffer: 5% (w/v) nonfat dry milk and 0.1% (v/v) Tween 20 in 1× PBS (140 mM NaCl, 2.7
mM KCl, 10 mM Na2HPO4, 1.8 mM KH2PO4, pH 7.3)
Wash buffer: 0.1% (v/v) Tween 20 in 1× PBS (as above)
Secondary antibody to detect the anti-GST antibody (such as anti-goat IgG HRP conjugate)
Appropriate membrane, such as Hybond ECL (for subsequent ECL detection) or Hybond P (for subsequent
ECL or ECL Plus detection)
1. Separate the protein samples by SDS-PAGE.
Although anti-GST antibody from GE Healthcare has been cross-adsorbed with E. coli proteins,
low levels of cross-reacting antibodies may remain. Samples of E. coli sonicates that do not
contain a recombinant pGEX plasmid and samples that contain the parental pGEX plasmid
should always be run as controls.
2. Transfer the separated proteins from the electrophoresis gel to the membrane.
Electrophoresis and protein transfer can be accomplished using a variety of equipment and reagents.
For further details, refer to the Protein Electrophoresis Technical Manual and Hybond ECL Instruction
Manual from GE Healthcare.
Blocking of membrane
1. Transfer the membrane onto which the proteins have been blotted into an appropriately sized container,
such as a Petri dish.
2. Add 50 to 200 ml of blocking/incubation buffer to the container.
3. Incubate for 1 to 16 h at ambient temperature with gentle shaking. Alternatively, block overnight at 4°C.
4. Decant and discard the buffer.
Longer incubation times with blocking/incubation buffer may reduce background signal.
Incubation of membrane blot with primary antibody

1. Prepare an appropriate dilution of anti-GST antibody with blocking/incubation buffer (e.g., 5 to 10 µl of
antibody in 50 ml of buffer).
5. Rinse the membrane twice with 20 to 30 ml of wash buffer to remove most of the unbound antibody.
6. Decant and discard the rinse buffers.
7. Wash the membrane with 20 to 30 ml of blocking/incubation or wash buffer for 10 to 60 min at ambient
temperature with gentle shaking.
8. Discard the buffer and repeat the wash from step 7.

Incubation of membrane blot with secondary antibody
1. Dilute an appropriate anti-goat secondary antibody with blocking/incubation buffer according to the
manufacturer’s recommendation.
5. Rinse twice with 20 to 30 ml of blocking/incubation or wash buffer to remove most of the unbound
antibody.
6. Decant and discard the rinse buffers.
7. Wash the membrane with 20 to 30 ml of blocking/incubation or wash buffer for 10 to 60 min at ambient
temperature with gentle shaking.
8. Discard the buffer and repeat the wash step using wash buffer.
Use wash buffer, not blocking/incubation buffer, for step 9. The protein in blocking/incubation
buffer would cause problems in the development step.
9. Develop the blot with a substrate that is appropriate for the conjugated secondary antibody.
Refer to GE Healthcare Application Note 18-1139-13 for further information on optimization of

antibody concentration for Western blotting.
ECL, ECL Plus, and ECL Advance detection systems require very little antibody to achieve a
sufficient sensitivity; therefore, the amount of antibody (primary and secondary) used in the
protocols can be minimized. Smaller quantities of antibody-buffer mixtures can be used by
scaling down the protocol and performing the incubations in sealable plastic bags.

GST Western Blotting Detection Kit
The GST Western Blotting Detection kit facilitates the detection of GST-tagged proteins on Western
blots using chemiluminescence. This method may be used for crude bacterial sonicates, column
eluates, or purified GST-tagged proteins. Unlike biochemical assays, this method is not dependent on
the functional activity of GST, which can be affected by folding of the tagged protein.
Components of GST Western Blotting Detection Kit

Anti-GST-HRP conjugate: Horseradish peroxidase (HRP) conjugated to goat anti-GST polyclonal antibody.
75 µl supplied in PBS containing 50% glycerol.
rGST positive control: recombinant Glutathione S-transferase. 10 µl supplied at 5 mg/ml protein
concentration in PBS.
ECL blocking agent: 20 g.
ECL detection reagents: detection reagent 1 (125 ml), detection reagent 2 (125 ml), sufficient for
2
2000 cm membrane.
Additional reagents required

Phosphate-buffered saline (PBS), pH 7.5: For 1 l, dissolve 11.5 g sodium hydrogen orthophosphate
anhydrous (80 mM), 2.96 g sodium dihydrogen orthophosphate (20 mM), and 5.84 g NaCl (100 mM) in
1 l of distilled water. Adjust pH.
Tris-buffered saline (TBS), pH 7.6: For 1 l add 20 ml 1 M Tris-HCl, pH 7.6 (20 mM) and 8 g NaCl to 1000 ml of
distilled water. Adjust pH.
PBS-Tween (PBST) and TBS-Tween (TBST) Dilute the required amount of Tween 20 in the corresponding
buffer. A 0.1% Tween 20 concentration is suitable for most blotting applications.
Do not use sodium azide as a bacteriocide as it inhibits horseradish peroxidase enzymes.
Gel electrophoresis
1. Perform SDS-PAGE according to standard techniques
2. Load 100 ng of the recombinant GST (included in the kit) as a positive control.
This conjugate has been used with both purified proteins and crude bacterial lysates. The anti-
GST antibody has been cross-absorbed against E. coli proteins. However, this process may not
remove all cross-reacting antibodies. We suggest that a sample of an E. coli lysate made from a
culture that does not contain a pGEX plasmid be run as a control.
Western blotting
1. Transfer the proteins onto a membrane using standard protocols.
2a. Prewet Hybond ECL in distilled water and equilibrate in transfer buffer for 5 to 10 min before blotting.
2b. Briefly immerse Hybond PVDF in 100% methanol then rinse in distilled water before equilibrating in
transfer buffer and blotting.
Nitrocellulose membrane is recommended with this protocol. However, PVDF membrane has
been shown to give comparable results.

Immunodetection
1. Block nonspecific binding sites by immersing the membrane in 5% ECL blocking agent in PBST or TBS for
1 h on a platform shaker at room temperature.
2. Briefly rinse the membrane in two changes of PBST or TBST wash buffer.
3. Dilute the anti-GST-HRP conjugate in PBST or TBST. A 1:5000 dilution has been found to be satisfactory for
many applications. Allow sufficient antibody solution to cover the membrane (at least 0.25 ml/cm2
membrane). Incubate the membrane in diluted conjugate for 1 h at room temperature on a platform
shaker.
4. Briefly rinse the membrane in two changes of PBST or TBST wash buffer. Wash the membrane in
2
> 4 ml/cm of wash buffer for 15 min at room temperature with gentle shaking.
5. Wash the membrane for 3 × 5 min with fresh changes of wash buffer at room temperature with gentle
shaking.
This protocol has been optimized to provide good signal-to-noise ratios, resulting in intense signal
and clean backgrounds. Users may find sensitivity is improved by increasing the concentration
of conjugate used; however, this may result in increased background noise.
ECL detection reagents are supplied with this kit. However, ECL Plus detection reagents have
also been used. When using ECL Plus reagents, increase conjugate dilution two-to-four-fold to
reduce background noise to an acceptable level.
ECL detection
1. Prepare the ECL detection reagents by mixing an equal volume of solution 1 with solution 2.
Allow sufficient volume to cover the membrane (at least 0.125 ml/cm2 is recommended).
Although the mixed reagents are stable for 1 h at room temperature, it is advisable to mix the
reagents immediately before use.
2. Drain the excess wash buffer from the washed membrane and place protein side up on a sheet of plastic
wrap or other suitable clean surface. Pipette the mixed reagents onto the membrane and incubate for 1 min.
3. Work quickly once the membrane has been exposed to detection reagents. Drain off excess reagents by
blotting the edge of the membrane on a tissue. Place the membrane on a fresh piece of plastic wrap,
protein side down. Wrap the membrane, taking care to gently smooth out any air bubbles.
4. Place the wrapped membrane protein side up in an X-ray film cassette.
5. Complete further stages in a dark room using red safe lights. Place a sheet of autoradiography film on
top of the membrane. Close the cassette and expose for 1 min.
6. Remove the film and replace with a second sheet of unexposed film. Develop the first piece of film
immediately. Dependent on the appearance of the first film, estimate the exposure time for the second
piece of film. This may vary from 5 min to 1 h.

SDS-PAGE with Coomassie blue or silver staining
SDS-PAGE is useful for monitoring tagged protein levels during expression and purification. Transformants
expressing the desired tagged protein are identified by the absence of the parental GST and by the
presence of a novel, larger tagged protein. Parental pGEX vectors produce a Mr 29 000 GST-tagged
protein containing amino acids coded for by the pGEX multiple cloning site.
Reagents required
6× SDS loading buffer: 0.35 M Tris-HCl, 10.28% (w/v) SDS, 36% (v/v) glycerol, 0.6 M dithiothreitol (or
5% 2-mercaptoethanol), 0.012% (w/v) bromophenol blue, pH 6.8. Store in 0.5 ml aliquots at -80°C.
Gel electrophoresis
1. Add 1 to 2 µl of 6× SDS loading buffer to 5 to 10 µl of supernatant from crude extracts, cell lysates, or
purified fractions, as appropriate.
2. Vortex briefly and heat for 5 min at 90°C to 100°C.
3. Centrifuge briefly, then load the samples onto an SDS-polyacrylamide gel.
4. Run the gel for the appropriate length of time and stain with Coomassie blue (Coomassie Blue R Tablets)
or silver (PlusOne Silver Staining Kit, Protein).
The percentage of acrylamide in the SDS-polyacrylamide gel should be selected based on the
expected molecular weight of the protein of interest (see Table 19).
Table 19. Selecting the appropriate gel composition for protein separation
Percent acrylamide in resolving gel Separation size range (Mr ×103)

Single percentage 5% 36–200
7.5% 24–200
10% 14–200
12.5% 14–100
15% 14–601
Gradient 5–15% 14–200
5–20% 10–200
10–20% 10–150
1The larger proteins fail to move significantly into the gel.

Troubleshooting of detection methods
The troubleshooting guide below addresses problems common to the majority of detection methods
as well as problems specific to a particular method. In the latter case, the relevant method is indicated.

Poor results with the The reaction rate is nonlinear. The reaction rate of the CDNB assay is
GST Detection Module linear provided that an A340 of ~ 0.8 is not
exceeded during the 5-min time course.
Plot initial results to verify that the reaction
rate is linear over the time course.
Adjust the amount of sample containing
the GST-tagged protein to maintain a linear
reaction rate.
The tagged protein is folded. The tagged protein may have inhibited the
correct folding of the GST moiety.
The GST-tagged proteins will thus show
very low activity with the CDNB assay.
Whether for this or for any other reason, if a
low absorbance is obtained using the CDNB
assay, a Western blot using anti-GST
antibody may reveal high levels of tagged
protein expression.
There is baseline drift . Under standard assay conditions at 22°C
and in the absence of GST, glutathione and
CDNB react spontaneously to form a
chemical moiety that produces a baseline
drift at ∆A340 /min of ~ 0.003 (or 0.015 in
5 min). Correct for baseline drift by blanking
the spectrophotometer with the blank
cuvette before each reading of the sample
cuvette. Alternatively, get slope directly
from the spectrophotometer software.
The slope will be the same as long as the
spontaneous reaction is limited.
Poor results with the GST Low absorbance is seen in Check that host cells were sufficiently
96-Well Detection Module the assay. induced, that the samples were sufficiently
lysed, and that inclusion bodies have not
been formed. (See Troubleshooting
purification methods).
Concentration of blocking buffer If clarified lysate is being tested, mix the
is inadequate. initial GST sample with 2× blocking buffer to
give a final concentration of 1× blocking
buffer.
There is poor day-to-day Verify that all incubation times are
reproducibility. consistent. GST capture incubation time
can be decreased with slightly reduced
signal, but do not incubate for less than
30 min. Every 15-min decrease in HRP/anti-
GST conjugate incubation time can
significantly reduce signal.

No signal in Western Proteins are not transferred Stain gel and membrane with total protein
blotting during Western blotting. stain to check transfer efficiency. Optimize
gel acrylamide concentration, time for
transfer, and current.
Ensure gel and membrane make proper
contact during blotting and are orientated
correctly with respect to the anode.
Check that excess temperatures are not
reached during electroblotting, producing
bubbles or membrane distortion.
Proteins are not retained on Assess transfer of proteins (as above). Use a
membrane. fresh supply of membrane.
There are problems with Ensure reagents are being used correctly.
detection reagents. Prepare reagents freshly each time. Store
reagents at correct temperature.
Weak signal in Protein transfer efficiency is poor. Check transfer efficiency as above.
Western blotting
Insufficient protein has been loaded. Load more protein on gel.
Exposure time is too short. Increase film exposure time; up to 1 h may
be required.
Conjugate concentration is too low. A 1:5000 dilution is recommended but a
more concentrated solution may be
required for some applications—try 1:1000.
Excessive diffuse signal Too much protein has been loaded. Reduce the amount of protein loaded.
in Western blotting
Conjugate concentration is too high. A 1:5000 dilution is recommended, but a
more dilute solution may be required for
some applications—try 1:10 000.
High backgrounds in Washing is inadequate. Ensure post conjugate washes are
Western blotting performed for a sufficient amount of time
with an adequate volume of wash buffer
(> 4 ml/cm2 membrane).
Blocking is inadequate. Check the blocking buffer has been made
correctly. Use freshly prepared blocking
buffer each time.
Increase the concentration of blocking
reagent—try 10%.
Use alternative blocking agent (e.g., 1% to
10% BSA, 0.5% to 3% gelatin.
Increase incubation time with blocking buffer.
Blotting equipment or buffers Clean equipment . Prepare fresh buffers.
are contaminated.
Conjugate concentration is too high. A 1:5000 dilution is recommended but
further dilution may be required for some
applications.
Multiple bands are seen Conjugate is binding nonspecifically Include a negative control of expression
in Western blotting to other proteins. host not containing expression vector to
determine nonspecific binding.

Removal of GST tag by enzymatic cleavage
Removal of the GST tag is often necessary to be able to perform functional or structural studies of the
target protein. Tagged proteins containing a PreScission Protease, thrombin, or Factor Xa recognition
site can be cleaved either while bound to Glutathione Sepharose or in solution after elution.
PreScission Protease itself has a GST tag and therefore will bind to Gluatahione Sepharose; it will
thus not co-elute and contaminate the cleaved target protein. Cleavage with PreScission Protease
is very specific, and maximum cleavage is obtained in the cold (the protein is most active at 4°C),
thus improving the stability of the target protein.
If thrombin or Factor Xa are used for cleavage of the tag, a convenient way to remove these
enzymes is to connect in series one GSTrap FF column and one HiTrap Benzamidine FF (high sub)
column. During the elution the cleaved product passes directly from the GSTrap into the HiTrap
Benzamidine FF (high sub). The cleaved target protein passes through the HiTrap Benzamidine
FF (high sub) column but the proteases bind. Thus in a single step the enzymes are removed and
a pure cleaved target protein is achieved (see Fig 68 on following page). Note, however, that
thrombin and Factor Xa may produce a less specific cleavage than PreScission Protease and
that sometimes the target protein can be fragmented itself.
Table 20. Approximate molecular weights for SDS-PAGE analysis.
Protease Molecular weight

PreScission Protease1 46 000
Bovine thrombin 37 000
Bovine Factor Xa 48 000
1 PreScission Protease is a fusion protein of glutathione S-transferase and human rhinovirus type 14 3C protease.
The amount of enzyme, temperature, and length of incubation required for complete digestion
varies according to the specific GST-tagged protein produced. Optimal conditions should always
be determined in pilot experiments.
If protease inhibitors (see Table 21) have been used in the lysis solution, they must be removed
prior to cleavage with PreScission Protease, thrombin, or Factor Xa. (The inhibitors will usually be
eluted in the flowthrough when sample is loaded onto a GSTrap column.)
Table 21. Inhibitors of the various proteases.
Enzyme Inhibitor
PreScission Protease 100 mM ZnCl2 (> 50% inhibition)
100 µM chymostatin
4 mM Pefabloc
Factor Xa and thrombin AEBSF, APMSF, antithrombin III, Antipain, α1-antitrypsin, aprotinin, chymostatin,
hirudin, leupeptin, PMSF
Factor Xa only Pefabloc FXa
Thrombin only Pefabloc TH Benzamidine

Cleavage of GST tag using PreScission Protease
Add cell lysate to Elute with Cleave eluted tagged
1 GST MultiTrap plate 3 reduced 4 protein with
or to prepacked glutathione PreScission
GST SpinTrap or Protease
GSTrap column
Off-column cleavage
2
Wash
On-column cleavage
Cleave tagged
3 protein with
PreScission
Protease
Fig 68A. Flow chart of the affinity purification procedure and PreScission Protease cleavage of GST-tagged proteins.
Cleavage of GST tag using thrombin or Factor Xa

Add cell lysate to Elute with Cleave eluted tagged
1 GST MultiTrap plate 3 reduced 4 protein with site-specific
or to prepacked glutathione protease (thrombin
GST SpinTrap or or Factor Xa)
GSTrap column
Off-column cleavage
2
Wash
On-column cleavage
Cleave tagged If using GSTrap FF, connect the GSTrap FF

3 protein with column directly to a HiTrap
site-specific Benzamidine FF (high sub) before
protease elution. Cleaved product passes
(thrombin directly from the GSTrap FF into
or Factor Xa) the HiTrap Benzamidine FF
(high sub). Samples are cleaved
and the protease removed
in a single step. HiTrap Benzamidine
FF (high sub)
Fig 68B. Flow chart of the affinity purification procedure and thrombin or Factor Xa cleavage of GST-tagged proteins.
Sepharose Glutathione Glutathione S-transferase Cloned protein
GST-tagged protein Thrombin or Factor Xa PreScission Protease

Add sample to
HiTrap Collect
5 6 GST MultiTrap, 7
Desalting eluate
GST SpinTrap,
column
or GSTrap FF
column Analyze protein, e.g.,
8 on SDS-PAGE or by
mass spectrometry
Analyze protein, e.g.,

5 on SDS-PAGE or by
mass spectrometry
Collect
4 flowthrough
Remove protease
if necessary,
Add sample to using HiTrap
5
HiTrap
6 GST MultiTrap, 7
Collect 9 Benzamidine FF
Desalting eluate
GST SpinTrap, (high sub)
column
or GSTrap
column

8 on SDS-PAGE or by
mass spectrometry

5 on SDS-PAGE or by
mass spectrometry
Collect
4 flowthrough Remove protease
if necessary,
Analyze protein, e.g., using HiTrap
4
Collect
flowthrough 5 on SDS-PAGE or by 6 Benzamidine FF
mass spectrometry (high sub)

Cleavage of tagged proteins is most commonly performed on milligram quantities of tagged protein
suitable for purification on GSTrap columns. Protocols that follow describe manual cleavage and
purification using a syringe and a 1-ml or 5-ml GSTrap column. The protocols can be adapted for use
with GST MultiTrap or GST SpinTrap columns to work at smaller scales.
For quick scale-up of purifications, two or three GSTrap columns can be connected in series (back
pressure will be higher). Further scaling-up is possible using GSTPrep FF 16/10 columns or columns
packed by the user. Protocols below are included for column or batch format using Glutathione
Sepharose 4 Fast Flow, but this medium can easily be replaced with Glutathione Sepharose High
Performance or Glutathione Sepharose 4B depending on what is the preferred media in the lab.
Cleavage and purification of GST-tagged protein bound to GSTrap FF

Recommended buffers
Binding buffer: PBS: 140 mM NaCl, 2.7 mM KCl, 10 mM Na2HPO4, 1.8 mM KH 2PO4, pH 7.3
For PreScission Protease cleavage:
Cleavage buffer: 50 mM Tris-HCl, 150 mM NaCl, 1 mM EDTA, 1 mM dithiothreitol (DTT), pH 7.0
PreScission Protease
For thrombin cleavage:
Cleavage buffer: PBS: 140 mM NaCl, 2.7 mM KCl, 10 mM Na2HPO4, 1.8 mM KH 2PO4, pH 7.3
Thrombin solution: Dissolve 500 units in 0.5 ml of PBS prechilled to 4°C. Swirl gently.
Store solution in small aliquots at -80°C to preserve activity.
For Factor Xa cleavage:
Cleavage buffer: 50 mM Tris-HCl, 150 mM NaCl, 1 mM CaCl2, pH 7.5
Factor Xa solution: Dissolve 400 units of Factor Xa in 4°C water to give a final solution of 1 unit/µl.
Swirl gently. Store solution in small aliquots at -80°C to preserve activity.
Purification and cleavage

The protocol below is an example optimized for 8 mg of target protein. It is worth estimating
how much target protein is applied to the column, as this allows one to minimize the amount of
protease added.
sample application.

7a. For PreScission Protease and Factor Xa, wash the column with 10 column volumes of cleavage buffer.
7b. For thrombin, proceed to step 8b.
7c. For Factor Xa, proceed to step 8c.
8a. Prepare the PreScission Protease mix:
–For GSTrap FF 1-ml columns, mix 80 µl (160 units) of PreScission Protease with 920 µl of PreScission
cleavage buffer at 5°C.
–For GSTrap FF 5-ml columns, mix 400 µl (800 units) of PreScission Protease with 4.6 ml of PreScission
cleavage buffer at 5°C.
8b. Prepare the thrombin mix:
–For GSTrap FF 1-ml columns, mix 80 µl (80 units) of thrombin solution with 920 µl of PBS.
–For GSTrap FF 5-ml columns, mix 400 µl (400 units) of thrombin solution with 4.6 ml of PBS.
8c. Prepare the Factor Xa mix:
–For GSTrap FF 1-ml columns, mix 80 µl (80 units) of Factor Xa solution with 920 µl of Factor Xa
cleavage buffer.
–For GSTrap FF 5-ml columns, mix 400 µl (400 units) of Factor Xa solution with 4.6 ml of Factor Xa
cleavage buffer.
9. Load the protease mix onto the column using a syringe and the connector supplied. Seal the column
with the top cap and the stopper supplied.
10a. For PreScission Protease, incubate the column at 5°C for 4 h.
10b. For thrombin and Factor Xa, incubate the column at room temperature (22°C to 25°C) for 2 to 16 h.
The incubation times are starting points and may need to be changed for an optimal yield of
cleaved target protein.
11. Fill a syringe with 3 ml (1-ml column) or 15 ml (5-ml column) of cleavage buffer. Remove the top cap and
stopper from the column and attach the syringe. Avoid introducing air into the column.
12. Begin elution of the cleaved target protein. Maintain flow rates of 1 to 2 ml/min (1-ml column) or
(5-ml column), and collect the eluate (0.5 to 1 ml/tube for 1-ml column, 1 to 2 ml/tube for 5-ml column).
For PreScission Protease: The eluate will contain the protein of interest, while the GST moiety of the
tagged protein and the PreScission Protease (also GST-tagged) will remain bound to the GSTrap
column. This means that the protein of interest will not be contaminated with protease and thus no
additional purification will be required to purify the target protein from the protease.
For thrombin and Factor Xa: The eluate will contain the protein of interest and thrombin or Factor Xa,
respectively, while the GST moiety of the tagged protein will remain bound to the GSTrap column. The
thrombin or Factor Xa can be removed from the protein of interest in one step using a HiTrap Benzamidine
FF (high sub) column in series after the GSTrap column. In this process, the cleaved, tagged protein and
thrombin or Factor Xa is washed from the GSTrap column onto the HiTrap Benzamidine FF (high sub)
column. This second column captures the thrombin or Factor Xa, thus enabling the collection of pure
protease-free protein in the eluent. Refer to the application on page 160 for an example of the purifica-
tion and on-column cleavage of GST-tagged SH2 domain using thrombin and GSTrap FF, with sample
clean-up accomplished using HiTrap Benzamidine FF (high sub) column in series with GSTrap FF.
See Appendix 2 for details on regenerating the GSTrap column for subsequent purifications.

1. Purification of human hippocalcin using GSTrap FF columns in series with on-column
cleavage by PreScission Protease
The gene for human hippocalcin, a member of the neurone-specific calcium-binding protein family,
was cloned into a pGEX vector containing a PreScission Protease site adjacent to the GST tag. The
expressed tagged protein was captured on a GSTrap FF 1-ml column. The column was then incubated
overnight at 4°C and for an additional 2 h at room temperature with PreScission Protease (which is
GST-tagged itself). Following on-column cleavage, a second GSTrap FF 1-ml column was placed in
series after the first to remove any PreScission Protease, uncleaved GST-tagged protein, or free GST tag
that could co-elute with the sample during the additional wash with binding buffer (Fig 69). For every
gram of wet E. coli cells, 10 mg of pure, untagged hippocalcin was obtained.
A)
Elution of
A 280
GSTrap FF
GST tag and PreScission Protease

fr.12
0.80
and uncleaved GST-tagged protein
PreScission Continued
Protease column wash
0.60
Column
wash
0.40
Hippocalcin
fr.2
fr.5
fr.6
0.20
0
0 10 20 30 40 ml
GSTrap FF
2× GSTrap FF
Sample: 2 ml clarified E. coli homogenate containing expressed GST-hippocalcin, Mr 43 000

Columns: 2× GSTrap FF 1-ml
Binding and wash buffer: 50 mM Tris-HCl, pH 8.0, 0.15 M NaCl, 1 mM CaCl2, 1 mM DTT, 10% glycerol
GST elution buffer: 20 mM reduced glutathione, 50 mM Tris-HCl, pH 8.0
System: ÄKTAprime
Protease treatment: 80 U/ml PreScission Protease overnight at 4°C and then 2 h at room temperature
B) 1 2 3 4 5 6 Lanes
Mr 1. Clarified E. coli homogenate containing expressed
97 000 GST-hippocalcin
66 000 2. Flowthrough (fraction 2)
GST-hippocalcin 3. GST-hippocalcin
45 000 4. Pure hippocalcin after on-column cleavage (fraction 5)
30 000 5. Same as lane 4, but fraction 6
20 100 GST 6. Eluted fraction from GSTrap FF containing PreScission
14 400 Protease and GST-tag released by cleavage (fraction 12)
hippocalcin (untagged)
Fig 69. Purification of human hippocalcin-GST-tagged protein with on-column cleavage and post-cleavage removal of PreScission
Protease using GSTrap FF columns. A) Chromatogram showing purification of hippocalcin. B) SDS-PAGE analysis of various sample
processing steps. ExcelGel SDS Gradient, 8–18%, Coomassie blue staining.

2. Automatic removal of the GST tag with PreScission protease
This example of automated tag removal uses ÄKTAxpress. All multistep purification protocols in ÄKTAxpress
can be combined with automated on-column tag cleavage. Tag cleavage is always performed on the
affinity column prior to further purification steps. When the cleaved protein has been eluted, the affinity
column is regenerated and affinity tag, tagged protease, and remaining uncleaved protein are collected
in a separate outlet. The procedure involves binding the tagged protein, injection of protease, incubation,
elution of cleaved protein, and collection in capillary loop(s), followed by further purification steps.
The example in Figure 70 shows purification results for a GST-tagged protein, GST-purα (Mr 61 600),
expressed in E. coli. The Mr of the cleaved product is 35 200. After harvest, cell lysis was performed by
sonication. The samples were clarified by centrifugation prior to sample loading.
Affinity chromatography (AC) and gel filtration (GF) were performed on ÄKTAxpress using columns as
indicated in the figure. The purity of each sample was analyzed by SDS-PAGE (Coomassie staining).
The reduced samples were applied on an ExcelGel SDS-polyacrylamide gel.
Sample: GST-purα, Mr 61 600 (cleaved product M r 35 200)
Columns: AC: GSTrap HP, 5 ml
GF: HiLoad 16/60 Superdex 75 pg
Cleavage conditions: 20 units of PreScission Protease/mg protein, 8 h incubation time in cold room
AC binding and 50 mM Tris-HCl, 150 mM NaCl, cleavage buffer: 1 mM EDTA, 1 mM DTT, pH 7.5
AC elution buffer: 50 mM Tris-HCl, 10 mM reduced glutathione, pH 8.0
GF buffer: 50 mM Tris-HCl, 150 mM NaCl, pH 7.5
A) B)
1 2 3 4 5
A 280
Cleaved protein Mr
mAU
97 000
66 000
2000
45 000
1500 Regeneration
30 000
20 100
46 mg
1000
14 400
500
Lanes
1. LMW marker
2. Start sample
3. Flowthrough
0 4. Purified cleaved GST-purα
200 250 300 ml 5. Reference: uncleaved GST-purα
AC GF
Fig 70. (A) Two-step protocol for automatic GST-tagged protein cleavage with PreScission Protease. (B) Analysis of the untagged target
protein after purification and GST-tagged cleavage on SDS-PAGE and Coomassie staining.

3. Purification and on-column cleavage of GST-tagged SH2 domain using thrombin and
GSTrap FF. Direct removal of thrombin using HiTrap Benzamidine FF (high sub) column in
series with GSTrap FF
The following application describes the purification of GST-SH2 (Mr 37 000) on a GSTrap FF-1 ml column,
followed by on-column cleavage with thrombin (Fig 71). After the thrombin incubation step, a HiTrap
Benzamidine FF (high sub) 1-ml column was placed in series below the GSTrap FF column. As the columns
were washed with binding buffer and later with high salt buffer, the cleaved SH2 tagged protein and
thrombin were washed from the GSTrap FF column onto the HiTrap Benzamidine FF (high sub) column.
Thrombin was captured by this second column, thus enabling the collection of pure thrombin-free
untagged target protein in the eluent (Fig 71A). Complete removal of thrombin was verified using the
chromogenic substrate S-2238 (Chromogenix, Haemochrom Diagnostica AB; supplier in US is DiaPharma)
for detection of thrombin activity (Fig 71B). This entire procedure could be completed in less than one day.
A) Lanes
Mr 1. LMW markers
97 000 2. Clarified E. coli homogenate containing SH2-GST-tagged
66 000 protein with a thrombin cleavage site
3. Flowthrough from GSTrap FF (fraction 2)
45 000 — SH2-GST
4. SH2 domain (GST tag cleaved off), washed out with
30 000 — GST binding buffer through both columns (fraction 6)
5. Same as lane 4 (fraction 7)
20 100 6. Same as lane 4 (fraction 8)
— SH2
14 400 7. Elution of thrombin from HiTrap Benzamidine FF (high sub)
8. Elution of GST tag and some noncleaved SH2-GST from
1 2 3 4 5 6 7 8 9 GSTrap FF (fraction 21)
9. Same as lane 8 (fraction 22)
High-salt Elution of HiTrap
B) buffer wash Benzamidine FF (high sub) Thrombin activity
A 280 Thrombin Elution of A 405
GSTrap FF
0.80
GST-tag 0.30
0.60
Thrombin Column
0.20
wash
0.40
0.20 Cleaved SH2 0.10

protein
fr.21
fr.22
fr.7
fr.2
fr.6
fr.14
fr.8
0 0
0 10 15 20 25 50 ml
A) A) A) B) A) A) GSTrap FF, 1 ml
B) HiTrap Benzamidine FF (high sub), 1 ml
B)
Sample: 2 ml clarified E. coli homogenate containing GST-SH2 (Mr 37 000)

with a thrombin cleavage site
Columns: GSTrap FF 1 ml and HiTrap Benzamidine FF (high sub) 1 ml
Binding buffer: 20 mM sodium phosphate, 0.15 M NaCl, pH 7.5
High salt wash buffer: 20 mM sodium phosphate, 1.0 M NaCl, pH 7.5
Benzamidine elution buffer: 20 mM p-aminobenzamidine in binding buffer
GST elution buffer: 20 mM reduced glutathione, 50 mM Tris, pH 8.0
System: ÄKTAprime
Protease treatment: 20 U/ml thrombin protease (GE Healthcare) for 2 h at room temperature
Thrombin activity: Measured at 405 nm using S-2238 (Chromogenix, Haemochrom Diagnostica AB;
supplier in US is DiaPharma) as substrate
Fig 71. Purification of GST-SH2 GST-tagged protein with on-column cleavage and post-cleavage removal of thrombin using GSTrap FF
and HiTrap Benzamidine FF (high sub) columns. (A) SDS-PAGE analysis of various sample processing steps. ExcelGel SDS Gradient 8–18%,
Coomassie blue staining. (B) Chromatogram and thrombin activity curve demonstrating all steps in the purification of the SH2 domain.

4. On-column cleavage of a GST-tagged protein using thrombin on a GSTrap FF column
To demonstrate the efficiency of on-column cleavage in conjunction with purification, a GST-tagged
protein containing the recognition sequence for thrombin was applied to GSTrap FF 1 ml. After washing,
the column was filled by syringe with 1 ml of thrombin solution (20 U/ml in PBS, pH 7.3) and sealed
using the supplied connectors. After incubation for 16 h at room temperature, the target protein minus
the GST moiety was eluted using PBS, pH 7.3, and the bound GST was subsequently eluted using elution
buffer (Fig 72). The cleavage reaction yield was 100%. Intact GST-tagged protein was not detected in
the eluate by SDS-PAGE and silver staining (see Fig 72C, lane 5).
A) B)
A 280 A 280 %
3.5 Elution
3.5
buffer
3.0 Wash 3.0 100
2.5 2.5
Incubation 80
2.0 16 h 2.0 Free
room temp. GST 60
1.5 1.5 Target
protein 40
1.0 1.0
0.5 0.5 20
0 0 0
5.0 10.0 15.0 min 2.0 4.0 6.0 8.0 10.0 12.0 min
Sample: 10 ml clarified cytoplasmic extract from E. coli Column: GSTrap FF 1-ml column after 16 h incubation
expressing a GST-tagged protein with thrombin
Column: GSTrap FF 1 ml Binding buffer: PBS, pH 7.3 (150 mM NaCl,
Binding buffer: PBS, pH 7.3 (150 mM NaCl, 20 mM phosphate buffer)
20 mM phosphate buffer) Elution buffer: 10 mM reduced glutathione, 50 mM Tris-HCl,
Flow rate: 1 ml/min pH 8.0
Chromatographic Flow rate: 1 ml/min
procedure: 4 column volumes (CV) binding buffer, Chromatographic
10 ml sample, 10 CV binding buffer, procedure: 8 column volumes (CV) binding buffer
fill column with 1 ml thrombin solution using (elution of cleaved target protein),
a syringe 5 CV elution buffer (elution of free GST and
System: ÄKTAexplorer 10 noncleaved GST-tagged protein),
5 CV binding buffer
C)
Mr
97 000
66 000
45 000 1. LMW
2. Cytoplasmic extract of E. coli expressing GST-tagged
30 000 protein, 1 g cell paste/10 ml
3. GST-tagged protein eluted from GSTrap 1 ml
20 100
4. GST-tagged protein eluted from GSTrap 5 ml
14 400 5. GST-free target protein eluted from GSTrap 1 ml after
16 h thrombin cleavage
6. Free GST eluted from GSTrap 1 ml after thrombin cleavage
7. Thrombin solution (20 U/ml)
1 2 3 4 5 6 7 8 8. LMW
Fig 72. On-column thrombin cleavage of a GST-tagged protein. (A) Equilibration, sample application, and washing of a GST-tagged
protein on GSTrap FF 1-ml were performed using ÄKTAexplorer 10. After washing, the column was filled by syringe with 1 ml of thrombin
(20 U/ml) and incubated for 16 h at room temperature. (B) GST-free target protein was eluted using PBS, pH 7.3. GST was eluted using
10 mM reduced glutathione. The GST-free target protein fraction also contained a small amount of thrombin not detectable by SDS-PAGE
(lane 6). The thrombin can be removed using a HiTrap Benzamidine FF (high sub) column. (C) SDS-PAGE followed by silver staining.

Cleavage and purification of GST-tagged protein eluted from GSTrap FF
Recommended buffers
Binding buffer: PBS: 140 mM NaCl, 2.7 mM KCl, 10 mM Na2HPO4, 1.8 mM KH 2PO4, pH 7.3
Cleavage buffer: PBS: 140 mM NaCl, 2.7 mM KCl, 10 mM Na2HPO4, 1.8 mM KH 2PO4, pH 7.3
Factor Xa solution: Dissolve 400 units of Factor Xa in 4°C water to give a final solution of 1 unit/µl.
Swirl gently. Store solution in small aliquots at -80°C to preserve activity.

The protocol below is an example optimized for 8 mg of target protein. It is worth estimating
how much target protein is applied to the column, as this allows one to minimize the amount of
protease added.
sample application.
7. Elute the GST-tagged protein with 5 to 10 column volumes of elution buffer. Maintain flow rates of 1 to
2 ml/min (1-ml column) or 1 to 5 ml/min (5-ml column). Collect the eluate (0.5 to 1 ml/tube for 1-ml
column, 1 to 2 ml/tube for 5-ml column). Pool fractions containing the GST-tagged protein (monitored by
UV absorption at A280).
8. Remove the free reduced glutathione from the eluate using a quick buffer exchange on HiTrap Desalting
or HiPrep 26/10 Desalting column, depending on the sample volume.
9a. For PreScission Protease, add 1 µl (2 units) of PreScission Protease for each 100 µg of tagged protein in
the buffer-exchanged eluate.

9b. For thrombin and Factor Xa, add 10 µl (10 units) of thrombin or Factor Xa solution for each mg of tagged
protein in the buffer-exchanged eluate.
10a. For PreScission Protease, incubate at 5°C for 4 h.
10b. For thrombin and Factor Xa, incubate at room temperature (22°C to 25°C) for 2 to 16 h.
The incubation times are starting points and may need to be changed for an optimal yield of
cleaved target protein.
11. Once digestion is complete, apply the sample to an equilibrated GSTrap FF column as described above
(steps 1 to 7) to remove the GST moiety of the tagged protein.
tagged protein and the PreScission Protease will remain bound to the GSTrap column. This means that
the protein of interest will not be contaminated with protease and thus no additional purification will be
required to purify the target protein from the protease.
respectively, while the GST moiety of the tagged protein will remain bound to the GSTrap column. The
thrombin or Factor Xa can be removed from the protein of interest in one step using a HiTrap Benzamidine
FF (high sub) column in series after the GSTrap column. In this process, the cleaved, tagged protein and
thrombin or Factor Xa is washed from the GSTrap column onto the HiTrap Benzamidine FF (high sub)
column. This second column captures the thrombin or Factor Xa, thus enabling the collection of pure
protease-free protein in the eluent.
See Appendix 2 for details on regenerating the GSTrap column for subsequent purifications.
Cleavage and purification of GST-tagged protein bound to Glutathione

Sepharose in batch mode
Recommended buffers
Glutathione Sepharose High Performance, Glutathione Sepharose 4 Fast Flow, and Glutathione
Sepharose 4B can all be used for cleavage and purification of GST-tagged proteins in batch.
Binding buffer: PBS: 140 mM NaCl, 2.7 mM KCl, 10 mM Na2HPO4, 1.8 mM KH2PO4, pH 7.3
Cleavage buffer: PBS: 140 mM NaCl, 2.7 mM KCl, 10 mM Na2HPO4, 1.8 mM KH2PO4, pH 7.3
Factor Xa solution: Dissolve 400 units of Factor Xa in 4°C distilled water to give a final solution of
1 unit/µl. Swirl gently. Store solution in small aliquots at -80°C to preserve activity.

Preparation of Glutathione Sepharose media and binding of protein
Glutathione Sepharose media are supplied in 20% ethanol. The media are used at a final slurry
concentration of 50%.
1. Determine the bed volume of Glutathione Sepharose medium required for your purification.
2. Gently shake the bottle to resuspend the slurry.
3. Use a pipette or measuring cylinder to remove sufficient slurry for use and transfer to an appropriate
container/tube.
5. Wash the Glutathione Sepharose media by adding 5 ml of PBS per 1 ml of 50% slurry.
Glutathione Sepharose media must be thoroughly washed with PBS to remove the ethanol
storage solution because residual ethanol may interfere with subsequent procedures.
7. Repeat steps 5 and 6 once for a total of two washes.
8. Add the cell lysate to the prepared Glutathione Sepharose medium and incubate for at least 30 min at
room temperature, using gentle agitation such as end-over-end rotation.

Assume 8 mg GST-tagged protein bound per ml medium.
1. Wash the tagged-protein-bound Glutathione Sepharose medium with 10 bed volumes of cleavage buffer.
Bed volume is equal to 0.5× the volume of the 50% Glutathione Sepharose slurry used.
2a. Prepare the PreScission Protease mix:
For each ml of Glutathione Sepharose bed volume, prepare a mixture of 80 µl (160 units) of PreScission
Protease and 920 µl of cleavage buffer at 5°C.
2b. Prepare the thrombin mix:
For each ml of Glutathione Sepharose bed volume, prepare a mixture of 80 µl (80 units) of thrombin and
920 µl of cleavage buffer.
2c. Prepare the Factor Xa mix:
For each ml of Glutathione Sepharose bed volume, prepare a mixture of 80 µl (80 units) of Factor Xa and
920 µl of cleavage buffer.
3. Add the protease mixture to the Glutathione Sepharose. Gently shake or rotate the suspension end-over-end.
4a. For PreScission Protease, incubate at 5°C for 4 h.
4b. For thrombin or Factor Xa, incubate at room temperature (22°C to 25°C) for 2 to 16 h.
The incubation times in steps 4a and 4b are starting points and may need to be changed for an
optimal yield of cleaved target protein.
5. Following incubation, wash out the untagged protein with approximately three bed volumes of cleavage
buffer. Centrifuge the suspension at 500 × g for 5 min to pellet the Glutathione Sepharose. Carefully
transfer the eluate to a tube.
tagged protein and the PreScission Protease will remain bound to the Glutathione Sepharose. This
means that the protein of interest will not be contaminated with protease and thus no additional
purification will be required to purify the target protein from the protease.

respectively, while the GST moiety of the tagged protein will remain bound to the Glutathione Sepharose.
The thrombin or Factor Xa can be removed from the protein of interest using HiTrap Benzamidine FF
(high sub). This column captures the thrombin or Factor Xa, thus enabling the collection of pure
protease-free protein in the eluent.
Removal of thrombin and Factor Xa using HiTrap Benzamidine FF (high sub)

Reagents required
Binding buffer: 0.05 M Tris-HCl, 0.5 M NaCl, pH 7.4
Elution buffer alternatives for eluting the protease:
0.05 M glycine-HCl, pH 3.0
10 mM HCl, 0.5 M NaCl, pH 2.0
20 mM p-Aminobenzamidine in binding buffer (competitive elution)
8 M urea or 6 M guanidine hydrochloride (denaturing solutions)
Recommended flow rates are 1 ml/min (1-ml column) or 5 ml/min (5-ml column).
1. Fill the pump tubing or syringe with distilled water. Connect the column to the syringe, using the connector
supplied, or to the pump tubing. Avoid introducing air into the column.
2. Remove the snap-off end.
3. Wash the column with five column volumes of distilled water to remove the storage buffer (0.05 M acetate
buffer, pH 4, containing 20% ethanol).
4. Equilibrate the column with five column volumes of binding buffer.
5. Apply the sample using a syringe fitted to the Luer connector or by pumping it onto the column.
Recommended flow rates for sample application are 1 ml/min for 1-ml column and 5 ml/min for 5-ml
column. Collect the flowthrough and reserve. It contains the protease-depleted material to be saved.
Apply a small volume of extra binding buffer to collect all desired material from the column.
6. Wash the column with 5 to 10 column volumes of binding buffer, collecting fractions (0.5 to 1 ml fractions
for 1-ml column and 1 to 3 ml fractions for 5-ml column) until no material appears in the effluent (monitored
by UV absorption at 280 nm).
7. Pool fractions from flowthrough and/or wash that contain the thrombin or Factor Xa free material
(monitored by UV absorption 280 nm).
8. For reuse of column, elute the bound protease with 5 to 10 column volumes of the elution buffer of choice.
If the eluted thrombin or Factor Xa is to be retained for reuse, buffer-exchange the fractions containing
the protease using HiTrap Desalting or PD-10 Desalting column. If a low pH elution buffer has been used,
collect fractions in neutralization buffer.
9. After all protease has been eluted, wash the column with binding buffer so it is ready for reuse.
If using low pH elution, collect protease fractions into neutralization buffer (60 to 200 µl of
1 M Tris-HCl, pH 9.0 per ml fraction collected), so that the final pH of the fractions will be
approximately neutral.
Thrombin activity can be followed by taking aliquots of the fractions and measuring at 405 nm
using S-2238 (Chromogenix, Haemochrom Diagnostica AB; supplier in US is DiaPharma) as
substrate.

Troubleshooting of cleavage methods
The troubleshooting guide below addresses problems common to the majority of cleavage methods as
well as problems specific to a particular method. In the latter case, the relevant method is indicated.
Situation Possible cause Remedy

GST-tagged proteins The ratios of PreScission Protease, Check the amount of tagged protein in
are not cleaved thrombin, or Factor Xa to GST-tagged the digest. Note that the capacity of the
completely. protein are not optimal. Glutathione Sepharose media for GST is
~ 10 mg/ml of medium for Glutathione
Sepharose High Performance and
Glutathione Sepharose 4 Fast Flow and
~ 5 mg/ml for Glutathione Sepharose 4B.
In most purifications, however, the medium
is not saturated with tagged protein. Verify
that the correct ratios of enzyme to protein
are used and adjust as necessary. For
PreScission Protease and thrombin, use at
least 10 units/mg of tagged protein. For
Factor Xa, use an amount equivalent to at
least 1% (w/w) of the weight of tagged
protein. For some tagged proteins, up to
5% Factor Xa can be used. The optimal
amount must be determined empirically.
In some cases, optimal results have been
achieved with a tagged protein concentration
of 1 mg/ml. The addition of = 0.5% SDS (w/v)
to the reaction buffer can significantly
improve Factor Xa cleavage with some
tagged proteins. Various concentrations of
SDS should be tested to determine the
optimal concentration.
The incubation time and/or enzyme Increase the incubation time for the
concentration is not sufficient for cleavage reaction. Increasing the reaction
complete cleavage of the protein time to 20 h or more should improve
from the GST tag. cleavage as long as the tagged protein is
not degraded by the extended incubation
period. Alternatively, try increasing the
amount of enzyme used for cleavage.
Specific cleavage sites for the Verify the presence of specific enzyme
proteases have been altered during cleavage sites. Check the DNA sequence of
cloning of the tagged protein. the construct and compare it with a known
sequence to verify that the cleavage sites
have not been altered.
The presence of cleavage enzyme Remove any enzyme inhibitors that may
inhibitors is interfering with the interfere with the cleavage reaction. Prior to
cleavage reaction. cleavage with PreScission Protease, buffer
exchange or dialyze the tagged protein
against 50 mM Tris-HCl, 150 mM NaCl,
1 mM EDTA, 1 mM DTT, pH 7.5. Prior to
cleavage with Factor Xa, buffer exchange
the tagged protein on HiTrap Desalting, a
PD-10 column, or HiPrep 26/10 Desalting,
depending on the sample volume, or dialyze
against 50 mM Tris-HCl, 150 mM NaCl,
1 mM CaCl2, pH 7.5.

Factor Xa is not properly activated. Activate Factor Xa with Russell´s viper
venom to generate functional enzyme.
For activation of Factor Xa, incubate
Russell´s viper venom with Factor Xa at a
ratio of 1% in 8 mM Tris-HCl, 70 mM NaCl,
8 mM CaCl2, pH 8.0. Incubate at 37°C for
5 min. Factor Xa from GE Healthcare has
been preactivated by this procedure.
The first amino acid after the Factor Check the sequence of the tagged protein
Xa recognition sequence is Arg or Pro. to verify that the first three nucleotides after
the Factor Xa recognition sequence do not
code for Arg or Pro.
Multiple bands are Proteolysis is occurring in the host Determine when the extra bands appear.
observed after bacteria prior to the cleavage Verify that additional bands are not present
electrophoresis/Western reaction. prior to PreScission Protease, thrombin, or
blotting analysis of the Factor Xa cleavage.
cleaved target protein
The tagged protein itself contains Check the sequence of the tagged protein
recognition sequences for to determine if it contains recognition
PreScission Protease, thrombin, sequences for the cleavage enzymes.
or Factor Xa.
The tagged partner is Glutathione Sepharose may have Pass the sample over a new GSTrap column
contaminated with been saturated with GST-tagged or fresh Glutathione Sepharose to remove
protease after protein during purification. residual PreScission protease, or over a
purification HiTrap Benzamidine (high sub) column in the
case of thrombin or Factor Xa.

Chapter 6
Simple purification of other recombinant or
native proteins
Numerous products are available, as discussed in previous chapters, that use affinity chromatography
to isolate and purify a specific histidine- or GST-tagged protein. However, many other tagged and
untagged proteins can also be isolated to a satisfactory degree of purity by a single-step purification
using affinity chromatography. In fact, single-step purification saves time (personnel and equipment)
and reduces both the risk of denaturation of the target protein and the loss of essential molecules that
are weakly attached to the protein. For high-throughput purification platforms, the need for additional
purification steps will increase the complexity of the task, and parallel formats become possible.
Fig 73. Single-step purification using specific affinity chromatography.
Affinity chromatography isolates a specific protein or a group of proteins with similar characteristics.
The technique separates proteins on the basis of a reversible interaction between the protein(s) and a
specific ligand attached to a chromatographic matrix. Whenever a suitable ligand is available for the
protein(s) of interest, a single affinity purification step offers high selectivity, hence high resolution, and
usually high capacity for the target protein(s). The basic principles of affinity chromatography are
outlined in Appendix 9.
Ready-to-use affinity purification columns

Table 22 shows the applications for which affinity purification with HiTrap and HiPrep columns are
already available. All columns are supplied with a detailed protocol that outlines the buffers and steps
required for optimal results. If higher binding capacity is needed, for larger-scale work, HiTrap columns
can be linked together in series to increase the capacity of a single purification step. Media are also
available for packing larger columns.
Table 22. Ready-to-use HiTrap and HiPrep columns for affinity purification.
Product Application Approx. binding Average pH

capacity particle stability
(mg/ml medium) size (long term)
MabSelect™ IgG, IgG subclasses, monoclonal 30 mg human IgG 85 µm 3–10
MabSelect SuRe™ IgG, IgG subclasses, monoclonal 30 mg human IgG 85 µm 3–13
MabSelect Xtra™ IgG, IgG subclasses, monoclonal 40 mg human IgG 75 µm 3–10
rProtein A FF IgG, IgG subclasses, human IgG 50 mg human IgG 90 µm 3–10
Protein A HP IgG, IgG subclasses, human IgG 20 mg human IgG 34 µm 3–9
Protein G HP IgG, rat IgG, mouse IgG1 25 mg human IgG 34 µm 3–9
IgM Purification HP Monoclonal IgM from hybridoma 5 mg human IgM 34 µm 3–11
supernatant
IgY Purification HP IgY from egg yolk 20 mg pure IgY 34 µm 3–11

Table 22. Ready-to-use HiTrap and HiPrep columns for affinity purification (continued).
Product Application Approx. binding Average pH

capacity particle stability
(mg/ml medium) size (long term)
Heparin HP Antithrombin III and other coagulation 3 mg AT III (bovine) 34 µm 5–10
factors, lipoprotein, lipases, DNA binding
proteins, protein synthesis factors
Blue HP Albumin, nucleotide-requiring 20 mg human albumin 34 µm 4–12
enzymes, coagulation factors
HisTrap HP Optimized high-performance At least 40 mg 34 µm 3–121
purification of histidine-tagged histidine-tagged protein
proteins
HisTrap FF Optimized purification of 40 mg histidine-tagged 90 µm 3–121
histidine-tagged proteins protein
HisPrep FF 16/10 Larger-scale, optimized purification 40 mg histidine-tagged 90 µm 3–121
of histidine-tagged proteins protein
HisTrap FF crude Optimized purification of 40 mg histidine-tagged 90 µm 3–121
histidine-tagged proteins. protein
Optimized for direct purification
from crude cell lysates.
IMAC HP Purification of proteins and peptides 40 mg histidine-tagged 34 µm 3–122
with exposed histidine groups, protein when charged
histidine-tagged proteins. with Ni2+. Metal ion and
Uncharged medium. protein dependent.
IMAC FF Purification of proteins and peptides 40 mg histidine-tagged 90 µm 3–122
with exposed histidine groups, protein when charged
histidine-tagged proteins. with Ni2+. Metal ion and
Uncharged medium. protein dependent.
HiPrep IMAC FF Larger-scale, optimized purification 40 mg histidine-tagged 90 µm 3–12 2
16/10 of proteins and peptides with protein when charged
exposed histidine groups, with Ni2+. Metal ion and
histidine-tagged proteins. protein dependent.
Uncharged medium.
Chelating HP Purification of proteins and peptides 23 µmol Cu2+ 34 µm 3–13
with exposed histidine groups,
histidine-tagged proteins.
Uncharged medium.
NHS-activated HP Coupling of own specific ligands 34 µm 3–12
via primary amino groups3
Streptavidin HP Biotinylated molecules, > 300 nmol biotin 34 µm 4–9
biotin-tagged proteins
GSTrap 4B GST-tagged proteins, other > 5 mg horse liver GST 90 µm 4–13
glutathione S-transferases, or
glutathione-dependent proteins
GSTrap HP GST-tagged proteins, other 10 mg recombinant 34 µm 3–12
glutathione S-transferases, or GST
glutathione-dependent proteins.
High-performance purifications.
GSTrap FF GST-tagged proteins, other 10 mg GST, 90 µm 3–12
glutathione S-transferases, or 11 mg GST-tagged
glutathione-dependent proteins protein, Mr 43 000
GSTPrep FF 16/10 Larger-scale purification of 10 mg GST, 90 µm 3–12
GST-tagged proteins, other 11 mg GST-tagged
glutathione S-transferases, or protein, Mr 43 000
glutathione-dependent proteins
Benzamidine FF Removal and/or purification of > 35 mg trypsin 90 µm 2–8
(high sub) serine proteases
1 Ni2+-stripped medium.
2 Uncharged medium.
3 The medium is pre-activated and a suitable ligand must be coupled to obtain an affinity medium.

Making a specific purification column
In cases when a ready-made affinity medium is unavailable, it may be considered worthwhile to develop
a “home-made” affinity purification column, for example, when a specific recombinant protein needs
to be prepared efficiently on a regular basis.
The ligand must be prepared, for example, by raising antibodies, tested for affinity to the target
protein, and purified before immobilized to a chromatographic matrix. For further details on general
purification strategies for proteins see Chapter 7 and the Protein Purification Handbook from
GE Healthcare. A detailed account of the principles of affinity chromatography can be found in the
Affinity Chromatography, Principles and Methods Handbook also available from GE Healthcare.
Use of HiTrap NHS-activated HP for simple preparation of an affinity purification column

NHS-activated Sepharose High Performance is a chromatographic matrix specifically designed for
the covalent coupling of ligands containing primary amino groups. This is the most common method
for coupling of proteins to chromatographic media. The matrix is based on highly cross-linked agarose
beads with 10-atom spacer arms attached to the matrix by epichlorohydrine and activated by
N-hydroxysuccinimide. The substitution level is ~10 µmol NHS-groups/ml medium. Nonspecific adsorption
of proteins (which can reduce binding capacity of the target protein) is negligible due to the excellent
hydrophilic properties of the base matrix.
The protocol below describes the preparation of an affinity medium using prepacked HiTrap NHS-
activated HP column and is generally applicable to all NHS-activated Sepharose products.
Optimal binding and elution conditions for purification of the target protein must be determined
separately for each ligand.
The activated matrix is supplied in 100% isopropanol to preserve stability prior to coupling.
Do not replace the isopropanol until it is time to couple the ligand.
Buffer preparation
Acidification solution: 1 mM HCl (ice-cold)
Coupling buffer: 0.2 M NaHCO3, 0.5 M NaCl, pH 8.3
Use high-quality water and chemicals. Filtration through 0.45 µm filters is recommended.
Coupling within pH range 6.5 to 9, maximum yield is achieved at pH ~8.
Ligand and column preparation

1. Dissolve desired ligand in the coupling buffer to a concentration of 0.5 to 10 mg/ml (for protein ligands).
If needed, perform a buffer exchange using a desalting column (see Chapter 9). The optimal concentration
depends on the ligand. Optimal sample volume is equivalent to one column volume.
2. Remove top-cap and apply a drop of ice-cold 1 mM HCl to the top of the column to avoid air bubbles.
3. Connect the top of the column to the syringe or system.
4. Remove the snap-off end.

Ligand coupling
1. Wash out the isopropanol with 6 column volumes of ice-cold 1 mM HCl.
Do not use excessive flow rates (maximum recommended flow rates are 1 ml/min (equivalent to
approximately 30 drops/min when using a syringe) with HiTrap 1 ml and 5 ml/min (equivalent to
approximately 120 drops/min when using a syringe) with HiTrap 5 ml). The column contents can
be irreversibly compressed.
2. Immediately inject one column volume of ligand solution onto the column.
3. Seal the column. Leave for 15 to 30 min at 25°C (or 4 h at 4°C).
If larger volumes of ligand solution are used, recirculate the solution. For example, when using a
syringe, connect a second syringe to the outlet of the column and gently pump the solution
back and forth for 15 to 30 min or, if using a peristaltic pump, simply recirculate the sample
through the column.
If required, the coupling efficiency can be measured at this stage. These procedures are
included in the instructions supplied with each HiTrap NHS-activated HP column package.
Washing and deactivation

This procedure deactivates any excess active groups that have not coupled to the ligand and washes
out nonspecifically bound ligands.
Buffer A: 0.5 M ethanolamine, 0.5 M NaCl, pH 8.3
Buffer B: 0.1 M acetate, 0.5 M NaCl, pH 4
1. Inject 3 × 2 column volumes of buffer A.

2. Inject 3 × 2 column volumes of buffer B.
4. Seal and leave the column for 15 to 30 min.
8. Inject 2 to 5 column volumes of a buffer with neutral pH.
The column is now ready for use.
Store the column in storage solution optimized for the specific column.
The presence of primary amines in the reaction mixture will inhibit the coupling reaction.
Buffers (e.g., Tris) or additives must be avoided.

Purification
Optimal binding and elution conditions for purification of the target protein using a specific
column must be determined separately for each ligand. Literature references and textbooks
may offer good guidelines. Below is a general protocol that can be used initially.
Use double-distilled or deionized water and high-quality chemicals. We recommend passing the
eluent through a 0.45 µm filter.
Samples should be centrifuged immediately before use and/or filtered through a 0.45 µm filter.
If the sample is too viscous, dilute with binding buffer, increase lysis treatment (sonication,
homogenization), or add DNase/RNase to reduce the size of nucleic acid fragments. Sample
binding properties can be improved by adjusting the sample to the composition of the binding
buffer. Dilute in binding buffer or perform a buffer exchange using a desalting column (see
Chapter 9).
Prepare the column

Perform a blank run (use binding buffer instead of sample) to ensure that loosely bound ligand is
removed (see below).
1. Wash with 3 column volumes of binding buffer.
3. Equilibrate with 5 to 10 column volumes of binding buffer.
Purification
1. Apply sample. Optimal flow rate is dependent on the binding constant of the ligand, but a recommended
flow rate range is, for example, 0.2 to 1 ml/min on a HiTrap 1-ml column.
2. Wash with 5 to 10 column volumes of binding buffer, or until no material appears in the eluent.
Avoid excessive washing if the interaction between the protein of interest and the ligand is
weak, because this may decrease the yield.
3. Elute with 2 to 5 column volumes of elution buffer.
4. If required, purified fractions can be desalted and exchanged into the buffer of choice using prepacked
desalting columns (see Chapter 9).
Reequilibrate the column

Reequilibrate the column by washing with 10 column volumes of binding buffer.

Chapter 7
Multistep purification of tagged and untagged
recombinant proteins
Recombinant protein expression may allow production of large amounts of an affinity-tagged protein
so that a single purification step using affinity chromatography is sufficient to achieve the desired level
of purity. However, the purification obtained after a single step is frequently not sufficient, and affinity
tags may sometimes interfere with the post-purification use of the protein. In these instances, multistep
purification will be necessary.
A significant advantage when working with recombinant proteins is that there is often considerable
information available about the product (amino acid sequence, Mr, pI, functional properties) and
contaminants (the expression host may be well known). With this information, detection assays and
sample preparation and extraction procedures in place, a purification strategy of Capture, Intermediate
Purification, and Polishing (CIPP) can be applied (Figure 74). This strategy is used in both the pharmaceutical
industry and in the research laboratory to ensure faster method development, a shorter time to pure
product, and good economy.
This section gives a brief overview of the approach recommended for any multistep protein purification.
Appendix 9 provides useful background information describing the various techniques discussed
herein. The Protein Purification Handbook (from GE Healthcare) is recommended as a guide to planning
efficient and effective protein purification strategies.
Purity
Polishing
Achieve final
high level purity
Intermediate
purification
Remove bulk
Capture impurities
Isolate, concentrate
Preparation, and stabilize
extraction,
clarification
Step
Fig 74. Preparation and CIPP.
CIPP is applied as follows:

Imagine the purification has three phases—Capture, Intermediate Purification, and Polishing.
Each phase may include one or more purification steps.
Assign a specific objective to each step within the purification process.
The problem associated with a particular purification step will depend greatly upon the properties of
the starting material. Thus, the objective of a purification step will vary according to its position in the
process, that is, at the beginning for isolation of product from crude sample, in the middle for further
purification of partially purified sample, or at the end for final clean-up of an almost pure product.
In the capture phase the objectives are to isolate, concentrate, and stabilize the target product. The product
should be concentrated and transferred to an environment that will conserve potency/activity.
During the intermediate purification phase the objectives are to remove most of the bulk impurities,
such as other proteins and nucleic acids, endotoxins, and viruses.

In the polishing phase most impurities have already been removed except for trace amounts or closely
related substances. The objective is to achieve final purity by removing any remaining trace impurities
or closely related substances.
The optimal selection and combination of purification techniques for Capture, Intermediate
Purification, and Polishing is crucial for an efficient purification.
Selection and combination of purification techniques

Proteins are purified using techniques that separate according to differences in specific properties, as
shown in Table 23.
Table 23. Techniques for protein purification.
Protein property Chromatographic technique

Charge Ion exchange (IEX),
Chromatofocusing
Size Gel filtration (GF)
Hydrophobicity Hydrophobic interaction (HIC),
Reversed phase (RPC)
Biorecognition (ligand specificity) Affinity (AC)
Resolution
Speed Recovery
Capacity
Fig 75. Every chromatographic technique offers a balance between resolution, capacity, speed, and recovery.
Resolution is achieved by the selectivity of the technique and the ability of the chromatographic
medium to produce narrow peaks. In general, resolution is most difficult to achieve in the final stages
of purification when impurities and target protein are likely to have very similar properties.
Capacity, in the simple model shown, refers to the amount of target protein that can be loaded during
purification. In some cases the amount of sample that can be loaded may be limited by volume (as in
GF) or by large amounts of contaminants that also bind the column, rather than by the amount of the
target protein.
Speed is of the highest importance at the beginning of purification, because the protein has not yet
been stabilized.
Recovery becomes increasingly important as the purification proceeds because of the increased value
of the purified product. Recovery is reduced by destructive processes in the sample and unfavorable
conditions on the column.
Select a chromatographic technique to meet the objectives for the purification step.
Choose logical combinations of purification techniques based on the main benefits of the
technique and the condition of the sample at the beginning or end of each step.

Combine techniques that are orthogonal to each other, that is, that apply very different separation
mechanisms.
Keep in mind the interplay between “required purity” and “required yield.” In general, every
added purification step will increase purity and decrease yield (except for desalting).
A guide to the suitability of each purification technique for the stages in CIPP is shown in Table 24.
Table 24. Suitability of purification techniques for CIPP.
Technique Main features Capture Inter- Polishing Sample start Sample end
mediate condition condition
IEX high resolution +++ +++ +++ low ionic strength, high ionic strength or
high capacity sample volume pH change,
high speed not limiting concentrated
sample
HIC good resolution ++ +++ + high ionic strength, low ionic strength,
good capacity sample volume concentrated
high speed not limiting, sample
addition of salt
needed
AC high resolution +++ +++ ++ specific binding specific elution
high capacity conditions, conditions,
high speed sample volume concentrated
not limiting sample
GF high resolution + +++ limited sample buffer exchanged
using Superdex volume (< 5% total (if required),
column volume) diluted sample
and flow rate range
RPC high resolution + +++ sample volume in organic solvent,
usually not limiting, risk loss of
additives may be biological activity
required
Minimize sample handling between purification steps by combining techniques to avoid the
need for sample conditioning before the next step. The product should be eluted from the first
column in a buffer suitable for the start conditions required for the next technique (see Table 24).
HIC (which requires high salt to enhance binding to the media) is well-suited as the capture step
after ammonium sulfate precipitation and clarification. The salt concentration and the total
sample volume will be significantly reduced after elution from the HIC column. Dilution of the
fractionated sample or rapid buffer exchange using a desalting column will prepare it for the
next IEX or AC step.
GF is a nonbinding technique with limited volume capacity and is unaffected by buffer conditions.
Because of its mechanism of acting, the sample zone in GF is broadened during passage through
the column. Eluted material may sometimes thus need to be concentrated. GF is well suited for
use after any of the concentrating techniques (IEX, HIC, AC).
Selection of the final strategy will always depend upon specific sample properties and the required
level of purification. Logical combinations of techniques are shown in Figure 76.

Proteins with low solubility
SDS SDS Solubilizing agents

extraction extraction (urea, ethylene glycol
nonionic detergents)
GF HIC HIC
(in nonionic
detergent)
GF GF
Crude sample or sample in high salt concentration

Sample
clarification GF GF GF
desalt mode desalt mode desalt mode
Capture AC IEX HIC IEX

dilution may
be needed
Intermediate IEX HIC

purification
Polishing GF GF GF GF
or or
RPC RPC
Clear or very dilute samples
Capture AC IEX IEX Precipitation

(e.g., in high
ionic strength)
Intermediate HIC Resolubilize

purification
Polishing GF GF GF Treat as for

or or sample in high
RPC RPC salt concentration
Fig 76. Example of logical combinations of chromatographic steps.
For the capture step, select a technique that binds the target protein and as few contaminants
as possible. In some cases it may be advantageous to select a technique that does not bind the
target protein but rather binds contaminants whose removal is critical, for example, proteases
or major contaminants.
A sample is purified using a combination of techniques and alternative selectivities. For example, in an
IEX-HIC-GF strategy the capture step selects according to differences in charge (IEX), the intermediate
purification step according to differences in hydrophobicity (HIC), and the final polishing step according
to differences in size (GF). This orthogonality in separation mechanisms allows very powerful purification
protocols for recombinant proteins without tags as well as for naturally abundant proteins.
If nothing is known about the target protein, use IEX-HIC-GF. This combination of techniques can
be regarded as a standard protocol.
Consider the use of both anion and cation exchange chromatography to give different selectivities
within the same purification strategy. Also consider the order of the techniques, as this will often
make a great difference in purification.

IEX is a technique that offers different selectivities using either anion or cation exchangers. A target
protein may very well bind to both exchangers at the same pH; alternatively, the pH can be changed.
The pH can be modified to alter the charge characteristics of the sample components. It is therefore
possible to use IEX more than once in a purification strategy, for capture, intermediate purification, or
polishing. IEX can be used effectively both for rapid separation in low-resolution mode during capture,
and in high-resolution mode during polishing in the same purification scheme.
Consider RPC for a polishing step provided that the target protein can withstand the run
conditions and is not irreversibly bound or denatured by the matrix.
RPC separates proteins and peptides on the basis of hydrophobicity. RPC is a high-resolution technique,
requiring the use of organic solvents. The technique is widely used for purity check analyses when
recovery of activity and tertiary structure are not essential. Because many proteins are denatured by
organic solvents, the technique is not generally recommended for protein purification where recovery
of activity and return to a native tertiary structure may be compromised. However, in the polishing
phase, when the majority of protein impurities have been removed, RPC can be excellent, particularly
for small target proteins that are less commonly denatured by organic solvents.
CIPP does not mean that all strategies must have three purification steps. For example, capture and
intermediate purification may be achievable in a single step, as may intermediate purification and
polishing. Similarly, purity demands may be so low that a rapid capture step is sufficient to achieve the
desired result. For purification of therapeutic proteins a fourth or fifth purification step may be required
to fulfil the highest purity and safety demands. The number of steps used will always depend upon the
purity requirements and intended use for the protein.
The following example demonstrates the successful application of CIPP in the purification of a
recombinant protein.

1. Three-step purification of a recombinant enzyme using ÄKTAFPLC™ System
This example demonstrates one of the most common purification strategies used when high purity
levels are required: IEX for capture, HIC for intermediate purification, and GF for the polishing step.
The objective was to obtain highly purified deacetoxycephalosporin C synthase (DAOCS), an oxygen-
sensitive enzyme that had been produced by overexpression in soluble form in the cytoplasm of
E. coli bacteria.
A more detailed description of this work can be found in Application Note 18-1128-91.
Sample extraction and clarification

Cells were suspended in Tris-based lysis buffer, pH 7.5 and lysed using ultrasonication. Streptomycin
sulfate and polyethyleneimine were added to precipitate DNA. The extract was clarified by centrifugation.
EDTA, DTT, benzamidine-HCl, and PMSF were used in the lysis buffer to inhibit proteases and minimize
damage to the oxygen sensitive-enzyme. Keeping the sample on ice also reduced protease activity.
Capture
The capture step focused on the rapid removal of the most harmful contaminants from the relatively
unstable target protein. This, together with the calculated isoelectric point of DAOCS (pI = 4.8), led to
the selection of an anion exchange purification. A selection of anion exchange columns, including
those from the HiTrap IEX Selection Kit, was screened to find the optimal medium (results not shown).
Optimization of the capture step (in Fig 77) allowed the use of a step elution at high flow rate to speed
up the purification.
mS/cm Column: HiPrep 16/10 Q XL
mAU Sample: Clarified E. coli extract
80 Start buffer: 50 mM Tris-HCl, 1 mM EDTA, 2 mM DTT,
3000 0.2 M benzamidine-HCl, 0.2 mM PMSF, pH 7.5
Elution buffer: Binding buffer + 1.0 M NaCl
60 Flow: 10 ml/min (300 cm/h)
2000
40
1000
20
0 0
0 100 200 ml
Fig 77. Capture using IEX. The elution position of DAOCS is shaded.

Intermediate purification
HIC was selected because the separation principle is complementary to IEX and because a minimum
amount of sample conditioning was required. Hydrophobic properties are difficult to predict, and it is
always recommended to screen different media. After screening, RESOURCE ISO was selected on the
basis of the resolution achieved. In this intermediate step, shown in Figure 78, the maximum possible
speed for separation was sacrificed in order to achieve higher resolution and allow significant reduction
of impurities.
mAU Column: SOURCE™ 15ISO, packed in HR 16/10
column
400 Sample: DAOCS pool from HiPrep 16/10 Q XL
Start buffer: 1.6 M ammonium sulfate, 10% glycerol,
300 50 mM Tris-HCl, 1 mM EDTA, 2 mM DTT,
0.2 mM benzamidine-HCl, 0.2 mM PMSF,
200 pH 7.5
Elution buffer (B): 50 mM Tris-HCl, 10% glycerol, 1 mM EDTA,
2 mM DTT, 0.2 mM benzamidine-HCl,
100 0.2 mM PMSF, pH 7.5
Gradient: 0–16%B in 4 CV, 16-24%B in 8 CV,
24–35%B in 4 CV, 100%B in 4 CV
0
0 100 200 ml Flow: 5 ml/min (150 cm/h)
CV = column volume
Fig 78. Intermediate purification using HIC. The elution position of DAOCS is shaded.
Polishing
The main goal of the polishing step, shown in Figure 79, was to remove aggregates and minor
contaminants and transfer the purified sample into a buffer suitable for use in structural studies.
The final product was used successfully in X-ray diffraction studies. This data is presented in more
detail in a Nature paper from 1998 [Structure of a cephalosporin synthase. Valegard, K., Terwisscha
van Scheltinga, A.C., Lloyd, M., Hara, T., Ramaswamy, S., Perrakis, A., Thompson, A., Lee, H.J., Baldwin,
J.E., Schofield, C.J., Hajdu, J. and Andersson, I. Nature 394, 805–809 (1998)].
mAU Column: HiLoad 16/60 Superdex 75 prep grade

Sample: Concentrated DAOCS pool from
1000
SOURCE 15ISO
Sample volume: 3 ml
800
Buffer: 100 mM Tris-HCl, 1 mM EDTA, 2 mM DTT,
0.2 mM benzamidine-HCl, 0.2 mM PMSF,
600
pH 7.5
Flow: 1 ml/min (30 cm/h)
400
200
0
0 20 40 60 80 100 ml
Fig 79. Polishing step using gel filtration. The elution position of DAOCS is shaded.

2. Three-step purification of a recombinant phosphatase using ÄKTAprime plus
The objective of this application was to produce a pure phosphatase (rPhosphatase) with retained
biological activity. The phosphatase gene was overexpressed and the protein was produced in soluble
form in the cytoplasm of E. coli. Using the preprogrammed method templates of ÄKTAprime plus with
prepacked HiPrep and HiLoad columns ensured quick and easy method development. The purification
strategy consisted of a capture step by IEX chromatography, intermediate purification by HIC, and
polishing by GF. Active rPhosphatase (35 mg) was purified within 8 h.
A more detailed description of this work can be found in Application Note 18-1142-32.
Sample preparation and extraction

The E. coli cells were suspended in lysis buffer, 1 g cells to every 10 ml lysis buffer (50 mM Tris-HCl,
1 mM EDTA, 2 mM DTT, pH 7.4). The suspended cells were lysed by ultrasonication, 6 × 20 bursts with
60 seconds cooling between each burst. DNA was removed by precipitation with 1% w/v streptomycin
sulfate. The sample was clarified by centrifugation, 15 min at 22 000 × g, before it was applied to the
first chromatography column.
Capture
The main purpose of the capture step was to concentrate the rPhosphatase and remove most of the
contaminants. The ÄKTAprime plus system pump was used to apply 200 ml of the clarified extract,
diluted 1:2 with water, to a HiPrep 16/10 DEAE FF column. A preprogrammed method template for IEX
chromatography was used for the separation.
Fractions of the eluate were collected and analyzed with an enzyme immunoassay detecting alkaline
phosphatase activity at an absorbance of 405 nm. The purity of the fractions containing rPhosphatase
was determined by SDS-PAGE.
A280 A 405 Sample: 200 ml clarified E. coli extract ,
2.0 4.0 diluted 1:2 with water, pH 6.6
UV absorbance
Column: HiPrep 16/10 DEAE FF, Vt = 20 ml
conductivity
Start buffer: 25 mM Tris-HCl, pH 7.4, 10% glycerol,
3.0 1 mM EDTA, 2 mM DTT
Elution buffer (B): 1 M NaCl in start buffer
1.0 2.0 Run parameters: Equilibration 0% B 2 CV
Sample application
Wash 1 0% B 4 CV
1.0 Elution 0–50% B in 20 CV
50% B for 1 CV
Wash 2 100% B 2 CV
0 CV = column volume
300 500 700 ml
Fig 80. Capture step using ion exchange. The phosphatase activity is represented by the green bars (absorbance at 405 nm).
Intermediate purification
HIC was used for intermediate purification because of its compatibility with samples containing a high
salt concentration. The pooled fractions from the IEX column were purified on HiLoad 16/10 Phenyl
Sepharose High Performance, using a preprogrammed method template in ÄKTAprime plus. The
fractions containing rPhosphatase were pooled and concentrated to 10 ml on an Amicon™ 50 ml
stirred-cell using a Diaflow™ PM10 filter. Reducing the sample volume enables a smaller GF column to
be used for the final polishing step.

A280 A 405 Sample: 170 ml rPhosphatase containing pool from
3.0 HiPrep 16/10 DEAE FF in 1.6 M ammonium
2.0 UV absorbance sulfate, pH 7.0
conductivity
Column: HiLoad 16/10 Phenyl Sepharose HP, Vt = 20 ml
Start buffer: 25 mM Tris-HCl, pH 7.4 in1.4 M ammonium
2.0 sulfate, 1 mM EDTA, 2 mM DTT
Elution buffer (B): 25 mM Tris-HCl in 10% glycerol, 1 mM EDTA,
2 mM DTT, pH 7.4
1.0
1.0 Run parameters: Equilibration 0% B 2 CV
Sample application
Wash 1 0% B 3 CV
Elution 0–100% B in 20 CV
0 Wash 2 100% B 2 CV
0 200 400 600 ml CV = column volume
Fig 81. Intermediate purification step using hydrophobic interaction. The phosphatase activity is represented by the green bars
(absorbance at 405 nm).
Polishing
The final polishing step used a preprogrammed method template to run a GF separation on a HiLoad
16/60 Superdex 75 prep grade column. The purity of the fractions containing rPhosphatase was checked
with SDS-PAGE (Fig 82) and by mass spectrometry (results not shown).
A)
A280 A 405 Sample: 4 ml concentrated eluate, containing rPhosphatase
from the HiLoad 16/10 Phenyl Sepharose HP
2.0 4.0 Column: HiLoad 16/60 Superdex 75 pg, Vt = 120 ml
UV absorbance
Buffer: 25 mM Tris-HCl, 300 mM NaCl, 1 mM EDTA, 2 mM DTT,
pH 7.4
3.0 Flow: 0.5 ml/min (15 cm/h)
CV = column volume
1.0 2.0
1.0
Vo Vt
0
0 50 100 ml
B)
Lanes
1. E. coli extract
Mr
2. Eluate from the capture step (ion exchange chromatography)
97 000
3. Eluate from the intermediate step (hydrophobic interaction chromatography)
66 000
4. Eluate from the polishing step (gel filtration)
rPhosphatase
30 000
20 100
14 400
1 2 3 4 5
Fig 82. (A) Polishing step using gel filtration. The phosphatase activity is represented by the green bars (absorbance at 405 nm).
(B) Purity check by SDS-PAGE. The proteins were stained with Coomassie Brilliant Blue.

Chapter 8
Handling inclusion bodies
Recombinant proteins are most often expressed in the intracellular space, but expression can also be
controlled so that the protein is secreted into the periplasmic space or out into the culture medium.
While secretion is advantageous in terms of protein folding, solubility, and disulfide bonding, the yield
is generally much higher when using intracellular expression.
However, recombinant protein accumulated intracellularly is frequently deposited in the form of inclusion
bodies, insoluble aggregates of misfolded protein lacking biological activity. The recombinant protein
is often the major component of inclusion bodies. Therefore the presence of inclusion bodies can make
preliminary isolation steps very simple, although the isolation of proteins from inclusion bodies often
leads to difficulties with refolding and full recovery of biological activity. Table 25 summarizes the
advantages and disadvantages of working with recombinant products expressed as inclusion bodies.
Inclusion body formation frequently occurs when eukaryotic proteins are expressed in bacterial hosts.
Table 25. Advantages and disadvantages of inclusion bodies.
High expression levels can reduce fermentation costs. Steps to refold the protein shift difficulties and costs
downstream.
Expression is easily monitored by SDS-PAGE or Expression cannot be monitored directly by functional
immunoblotting and visually by microscopic assays.
analysis (inclusion bodies often can be observed
as dark particles in the bacterial cells).
Inclusion bodies can be isolated to high purity and Minor contaminants are often hydrophobic, poorly
used directly as antigen. soluble membrane proteins and cell wall fragments.
Tagged proteins are generally protected from Major contaminants are oligomers and misfolded or
proteolytic breakdown. proteolyzed forms of the protein that can be difficult
to separate.
Small tagged proteins present in inclusion bodies If the protein does not refold well, another expression
refold with good efficiency. system will be needed.
If the protein is expressed as inclusion bodies, there are several options to consider: optimize as much
as possible for soluble expression, accept the formation of inclusion bodies but develop strategies to
solubilize and refold the protein, try another expression host, or modify the plasmid construct.
Solubilization of inclusion bodies

The solubility of a recombinant protein can be made more favorable by modification of culture
conditions.
A variety of growth parameters can be investigated, either solely or in combination, that may provide
a good yield of nondegraded tagged protein in the soluble fraction. Steps to investigate include:
• Lowering the growth temperature to between 20°C and 30°C
• Increasing aeration
• Altering induction conditions
In general, induction at lower cell densities (A600 = 0.5) usually results in greater yields of the tagged
protein in a soluble form. However, in some cases it may be beneficial to grow the cells to a higher cell
density (> 1 A600 unit) for a shorter period of time, or simply to induce for a shorter period of time.

Growing the cells to a higher cell density and either omitting induction (for example, by IPTG) or reducing
the concentration of the inducing agent (for example, with IPTG, reducing the concentration to 0.1 mM)
leads to lower yields, but more of the tagged protein is likely to be obtained in an intact form.
If culture modifications do not significantly improve the yield of soluble tagged proteins, then
common denaturants such as 4 to 6 M guanidine hydrochloride, 4 to 8 M urea, detergents,
alkaline pH (> 9), organic solvents, or N-lauroyl-sarcosine can be used to solubilize inclusion
bodies.
For each denaturant the success of solubilization will be affected by the presence and concen-
tration of reducing agent, time, temperature, ionic strength, and the ratio of denaturant to
protein. Solubilized proteins can often be purified at this stage by using a separation technique
that is compatible with the presence of the denaturant. Purification and refolding can often be
combined in the same purification step, for example, by chromatographic on-column refolding.
Success of affinity purification in the presence of denaturing agents will depend on the nature
of the tagged protein. Denaturants such as guanidine hydrochloride, urea, Tween 20, CTAB, or
SDS have all been used, but it is important to test the chosen denaturant with the target protein
before introducing it into the solubilization strategy.
Refolding of solubilized recombinant proteins

Following solubilization, proteins must be properly refolded to regain function. Denaturing agents
must always be removed to allow refolding of the protein and formation of the correct intramolecular
associations. Critical parameters during refolding include pH, presence of reducing reagents, the speed
of denaturant removal, and the relative concentrations of host proteins and recombinant protein. Table 26
compares conventional methods for refolding of insoluble recombinant proteins with on-column
affinity purification and refolding.
Refolding usually requires extensive optimization. One should always consider other alternatives
(as mentioned earlier), for example, optimizing expression parameters, making a new construct,
or changing the expression host.
Table 26. Comparison of methods for protein refolding.
Refolding techniques Advantages/Disadvantages

Step dialysis Takes several days.
Uses large volumes of buffer.
Dilution into near neutral pH Simple technique. Gives extensive dilution, often several-hundred-fold.
Gel filtration Slow.
Requires a second column to be run.
Only small volumes can be processed per column.
On-column refolding Fast and simple.
No sample volume limitations.
Success varies and is dependent on the protein.

On-column refolding
Using a histidine-tagged protein enables the use of a simple, but efficient, purification and on-column
refolding procedure that produces soluble protein exhibiting the desired biological activity. The protocol
shown in Figure 83 has been used successfully for several different histidine-tagged proteins.
E. coli culture
Cell paste
Cell disruption
Centrifugation (5–10 000 g)
Pellet
Wash & centrifugation
Isolated inclusion bodies
Solubilization
Purification & refolding on

HisTrap column
Fig 83. General scheme for the extraction, solubilization, and refolding of (histidine)6-tagged proteins produced as inclusion bodies in
E. coli cells.
High concentrations of chaotropic agents (such as urea or guanidine hydrochloride) enhance the
binding of the histidine tag to immobilized divalent metal ions. Consequently, (histidine)6-tagged proteins
can be solubilized by chaotropic extraction and bound to Ni Sepharose. Removal of contaminating
proteins and refolding by exchange to nondenaturing buffer conditions can be performed before
elution of the protein from the column.
Once refolded, the protein may be purified further by other techniques (see Chapter 7) if a higher
degree of purity is required.

Application
Purification and on-column refolding of an insoluble histidine-tagged protein from
a 100 ml E. coli culture using HisTrap FF 1 ml with ÄKTAprime plus
This procedure uses a HisTrap FF 1-ml column but also can be used with a HisTrap HP 1-ml or a
HisTrap FF crude 1-ml column.
Preparing the buffers

Resuspension buffer: 20 mM Tris-HCl, pH 8.0

Isolation buffer: 2 M urea, 20 mM Tris-HCl, 0.5 M NaCl, 2% Triton-X 100, pH 8.0
Binding buffer (port A1): 6 M guanidine hydrochloride, 20 mM Tris-HCl, 0.5 M NaCl, 5 mM imidazole,
1 mM 2-mercaptoethanol, pH 8.0
Solubilization buffer (port A2): 6 M urea, 20 mM Tris-HCl, 0.5 M NaCl, 5 mM imidazole,
Elution buffer (port A3): 20 mM Tris-HCl, 0.5 M NaCl, 0.5 M imidazole, 1 mM 2-mercaptoethanol,
pH 8.0
Refolding buffer (port B): 20 mM Tris-HCl, 0.5 M NaCl, 5 mM imidazole, 1 mM 2-mercaptoethanol,
pH 8.0
Prepare at least 500 ml of each eluent .
Alternative binding buffers: 5 to 40 mM imidazole can be included in the binding buffer to reduce
nonspecific binding of non-histidine-tagged proteins. The concentration of imidazole is protein
dependent, and if the protein of interest elutes or does not bind at a certain imidazole concentration,
reduce the concentration.
Disruption, wash, and isolation of inclusion bodies

1. Resuspend the cell paste from 100 ml culture in 4 ml resuspension buffer.
2 Disrupt cells with sonication on ice (e.g., 4 × 10 sec).
3. Centrifuge at high speed for 10 min at 4°C.
4. Remove supernatant and resuspend pellet in 3 ml of cold isolation buffer. Sonicate as above.
5. Centrifuge at high speed for 10 min at 4°C.
6. Repeat steps 4 and 5.
At this stage the pellet material can be washed once in buffer lacking urea and stored frozen for
later processing.
Solubilization and sample preparation

1. Resuspend pellet in 5 ml of binding buffer.
2. Stir for 30 to 60 min at room temperature.
3. Centrifuge for 15 min at high speed, 4°C.
4. Remove any remaining particles by passing sample through a 0.45 µm filter.
The optimal concentration of 2-mercaptoethanol (0 to 20 mM) must be determined experimentally

for each individual protein.

If it has not been prepared as above, adjust the sample to the composition of binding buffer by
diluting in binding buffer or by buffer exchange using HiTrap Desalting or HiPrep 26/10 Desalting,
then pass the sample through a 0.45 µm filter.
Preparing the system

The requirement for linear gradient formation for refolding and elution makes the use of a
chromatography system essential.
This example uses ÄKTAprime plus. Once the system is prepared, the remaining steps (under
Selecting Application Template and starting the method) will be performed automatically.
1. Place each inlet tubing from port A (8-port valve) in eluents as given above and the tubing from port B
(2-port valve) in the elution buffer.
5. Connect a sample loop large enough for your sample between port 2 and 6 on the injection valve.
Use a syringe to manually fill the loop.

2. Use the arrow and OK buttons to move in the menu tree until you find On-Column Refolding HisTrap.
Set Sample Inj. Vol

Templates
(00.0 ml) 00.0

Press OK to start
On-Column Refolding
Run data displayed
HisTrap
%B
100
Refolding Elution
Priming
Reequilibration
Buffer wash
50 & Priming
Equili-
bration
Sample
2 10 30 60 20 20 17 Min
Total separation time = 160 min + sample application time
Fig 84. Theoretical gradient in On-column Refolding HisTrap Application Template.

AU280 Sample: Clarified homogenate of
%B
E. coli expressing histidine-
UV 280 nm 100
tagged protein
Programmed %B Column: HisTrap FF 1 ml
0.4
80 Binding buffer (A1): 6 M guanidine hydrochloride,
20 mM Tris-HCl, 0.5 M NaCl,
5 mM imidazole,
0.3 60 1mM 2-mercaptoethanol, pH 8.0
Solubilization buffer (port A2): 6 M urea, 20 mM Tris-HCl,
0.2 40 0.5 M NaCl, 5 mM imidazole,
1mM 2-mercaptoethanol, pH 8.0
Elution buffer (port A3): 20 mM Tris-HCl, 0.5 M NaCl,
0.1 20 0.5 M imidazole,
Refolding buffer (port B): 20 mM Tris-HCl, 0.5 M NaCl,
0 0 5 mM imidazole,
20 40 60 80 100 120 140 min 1 mM 2-mercaptoethanol, pH 8.0
Fig 85. Typical results for on-column refolding of a histidine-tagged protein.
Troubleshooting
Situation Possible cause Remedy
High back pressure Column clogged Clean the column according to instructions or replace it with a
fresh column. Make sure the sample has been centrifuged
and/or filtered through a 0.45 µm filter.
System clogged Replace the column with a piece of tubing. Check pressure.
If back pressure > 0.3 MPa, clean system according to manual.
No binding – Check that the correct column is used.
Check that the inlet tubing from each buffer is connected to
the correct inlet port.
Check that correct “method program” was selected.
Check that the composition and pH of the buffers are correct .
Check that the sample has been adjusted to binding buffer
conditions.
No elution – Check that the inlet tubing from each buffer is connected to
the correct inlet port.
Check that correct “method program” was selected.
Check that the composition and pH of the buffers are correct .
Use alternative elution conditions according to the column
instructions.
Check flow of buffer by looking for liquid coming from the
outlet of the system.

Chapter 9
Desalting columns provide a simple and fast method to remove salt or other small molecules from a
sample and exchange its buffer composition. The technique can also be used for adjusting to a higher
salt concentration or for removal of additives that are not needed in subsequent use of the material.
The columns are packed with Sephadex™ G-25, a gel filtration product that separates molecules on the
basis of size. Desalting provides several advantages over dialysis, which is generally a slow technique
that requires large volumes of buffer and carries a risk of losing material during handling or because of
proteolytic breakdown, aggregation, or nonspecific binding of samples to the dialysis membranes.
With desalting columns, in a single step the sample is desalted, transferred into a new buffer, and low
molecular weight materials are removed, all within minutes. The columns are also used for the rapid
removal of reagents to terminate a reaction.
Sample volumes up to 30% of the total volume of the desalting column can be processed. The high
speed and high capacity of the separation allows even large sample volumes to be processed rapidly
and efficiently. Sample concentration does not influence the separation as long as the concentration
of proteins does not exceed approximately 70 mg/ml when using normal aqueous buffers, and provided
that the protein is stable and soluble at the concentration used. Table 27 shows a selection guide for
prepacked, ready-to-use desalting and buffer exchange columns.
Table 27. Selection guide for desalting/buffer exchange columns.
Column Medium Loaded Eluted Dilution Operation

volume (ml) volume (ml) factor
NAP™-5 Sephadex G-25 0.1 0.5 5 gravity
DNA Grade
0.25 0.7 2.8 gravity
0.5 (max.) 1.0 2 gravity
NAP-10 Sephadex G-25 0.75 1.2 1.6 gravity
DNA Grade
1.0 (max.) 1.5 1.5 gravity
PD-10 Desalting Sephadex 1.5 3.5 1–1.5 gravity
G-25 Medium
2.0 3.5 1–1.3 gravity
2.5 (max.) 3.5 1–1.3 gravity
HiTrap Desalting Sephadex 0.25 1.0 4 syringe/pump
G-25 Superfine
0.5 1.5 3 syringe/pump
1.0 2.0 2 syringe/pump
1.5 (max.) 2.0 1.3 syringe/pump
2x HiTrap Desalting Sephadex 3.0 (max.) 4–5 1.3–1.7 syringe/pump
G-25 Superfine
3x HiTrap Desalting Sephadex 4.5 (max.) 6–7 1.3–1.7 syringe/pump
G-25 Superfine

Table 27. Selection guide for desalting/buffer exchange columns (continued).
Column Medium Loaded Eluted Dilution Operation

volume (ml) volume (ml) factor
HiPrep 26/10 Desalting Sephadex 10 10–15 1–1.5 pump
G-25 Fine
15 (max.) 15–20 1–1.3 pump
2x HiPrep 26/10 Desalting Sephadex 30 (max.) 30–40 1–1.3 pump
G-25 Fine
G-25 Fine
G-25 Fine
To desalt larger sample volumes:

Connect up to five HiTrap Desalting columns in series to increase the sample volume capacity, for
example, two columns, sample volume 3 ml; five columns, sample volume 7.5 ml.
Connect up to four HiPrep 26/10 Desalting columns in series to increase the sample volume capacity,
for example, two columns, sample volume 30 ml; four columns, sample volume 60 ml. Even with four
columns in series the sample can be processed in 20 to 30 minutes.
Buffer preparation:
For substances carrying charged groups, an eluent containing a buffer salt is recommended. A salt
concentration of at least 25 mM is recommended to prevent possible ionic interactions with the
medium. Sodium chloride is often used for this purpose. Often a buffer with 25 to 50 mM concentration
of the buffering substance is sufficient.
At salt concentrations above 1.0 M, hydrophobic substances may be retarded or bind to the medium.
At even higher salt concentrations [> 1.5 M (NH4)2SO4], the column packing shrinks.
Sample preparation:
The sample concentration does not influence the separation as long as the viscosity does not differ by
more than a factor of 1.5 from that of the buffer used. This corresponds to a maximum concentration
of 70 mg/ml for proteins or 5 mg/ml for high molecular weight polymers such as dextran, when
normal aqueous buffers are used.
The sample should be fully solubilized. Centrifuge or filter (0.45 µm filter) immediately before loading to
remove particulate material if necessary.
Protein solubility often depends on pH and/or ionic strength (salt concentration), and the exchange of
buffer may therefore result in precipitation of the protein. Also, protein activity can be lost if the
change of pH takes it outside of the range where the protein is active.
The protocols below describe desalting and buffer exchange using PD-10 Desalting, HiTrap Desalting,
and HiPrep 26/10 Desalting columns.

PD-10 Desalting
PD-10 Desalting columns are packed with Sephadex G-25 medium for group separation of high
(Mr > 5 000) from low molecular weight substances (Mr < 1 000) by desalting and buffer exchange. The
medium is held within two sintered polyethylene frits. The frits protect the medium from running dry
under gravitational buffer flow. The outlet to the column is sealed with a reusable cap.
Each column can process a sample volume up to 2.5 ml by gravity flow, and multiple samples can be
processed in parallel. PD-10 columns are recommended for use with bulk media for affordable and
versatile sample preparation.
The columns are packaged with a column stand—the PD-10 Desalting Workmate; LabMate buffer
reservoir is available for easy equilibration.
1. 2. 3. 4. 5.
Fig 86. Schematic of the method used with PD-10 Desalting columns. (1) Preparation of the column; (2) attachment of the LabMate
Buffer Reservoir, (3) column equilibration, (4) sample application, (5) elution and collection of sample.
1. Cut off bottom tip, remove top cap, and pour off excess liquid.
2. If available, mount the LabMate Buffer Reservoir on top of the PD-10 column and place the columns in
the PD-10 Desalting Workmate.
3. Equilibrate the column with approximately 25 ml of buffer. Discard the flowthrough (you can use the
plastic tray to collect the flowthrough).
4. Add sample of a total volume of 2.5 ml. If the sample is less than 2.5 ml, add buffer until the total volume
of 2.5 ml is achieved. Discard the flowthrough.
5. Elute with 3.5 ml of buffer and collect the flowthrough. A typical separation is shown in Figure 87.
Column: PD-10
Concentration
Albumin NaCl Sample: HSA, 25 mg in 2.5 ml 0.5 M NaCl

Equilibration: Distilled water
0 2 4 6 8 10 12
Elution volume (ml)
Fig 87. Removal of NaCl from albumin solution. A PD-10 Desalting column was equilibrated with distilled water. The sample contained
human serum albumin (25 mg) dissolved in 2.5 ml 0.5 M NaCl solution. A total of 23.8 mg albumin was recovered in 3.5 ml eluent
corresponding to a yield of 95.3% (between arrows). Initial total salt content of sample before desalting was 2.0%.

HiTrap Desalting
Fig 88. HiTrap Desalting allows efficient, easy-to-perform group separations with a syringe or pump.
HiTrap Desalting is a 5-ml column packed with the well-known size-exclusion medium Sephadex G-25
Superfine. The medium is based on cross-linked dextran beads that allow excellent resolution and high
flow rates. The fractionation range for globular proteins is between Mr 1 000 and 5 000, with an exclusion
limit of approximately Mr 5 000. This ensures group separations of proteins/peptides larger than
Mr 5 000 from molecules with a molecular weight less than Mr 1 000.
HiTrap Desalting can be used with aqueous solutions in the pH range 2 to 13. It is stable to all commonly
used buffers, solutions of urea (8 M), guanidine hydrochloride (6 M), and all nonionic and ionic deter-
gents. Lower alcohols (methanol, ethanol, propanol) may be used in the buffer or the sample, but we
recommend that the concentration be kept below 25 v/v%. Prolonged exposure (hours) to pH values
below 2 or above 13, or to oxidizing agents, should be avoided.
The recommended range of sample volumes is 0.1 to 1.5 ml when complete removal of low molecular
weight components is desired. The separation is not affected by the flow rate, in the range 1 to 10 ml/min.
The maximum recommended flow rate is 15 ml/min. The column cannot be opened or repacked.
Separations are easily performed with a syringe, a pump, or a chromatography system such as one of
the ÄKTAdesign systems. Up to five columns can be connected in series, allowing larger sample volumes
to be handled. Refer to Scaling up on page 197 for further discussion of these options.
Figure 89 shows a typical desalting and buffer exchange separation achieved using HiTrap Desalting
and monitored by following changes in UV absorption and conductivity.
Conductivity Column: HiTrap Desalting

A 280 mS/cm Sample: 2 mg/ml BSA in 50 mM sodium phosphate,
0.5 M NaCl, pH 7.0
0.8 50 Sample volume: 1.4 ml
NaCl
Buffer: 50 mM sodium phosphate, 0.15 M NaCl, pH 7.0
Flow rate: 10 ml/min
0.6 Detection: UV (280 nm, 5 mm cell) and conductivity
40
BSA
0.4
30
0.2
20
0.0
0 10 20 30 40 50 Time (s)
Fig 89. Highly efficient desalting in 30 seconds using HiTrap Desalting.
To avoid cross-contamination, use the column only with the same type of sample.

Column equilibration
1. Fill the syringe or pump tubing with buffer. Remove the stopper. To avoid introducing air into the column,
connect the column “drop to drop” to either the syringe (via the connector) or to the pump tubing.
3. Wash the column with 25 ml of buffer at 5 ml/min to completely remove the storage buffer, which
contains 20% ethanol. If air is trapped in the column, wash with degassed buffer until the air disappears.
Air introduced into the column by accident during sample application does not influence the separation.
Note: 5 ml/min corresponds to approximately 120 drops/min when using a HiTrap 5-ml column.
Manual desalting using a syringe

1. To operate the column with a syringe, connect the syringe to the column using the supplied Luer connector.
2. Equilibrate the column; see above, Column equilibration.
3. Apply the sample using a 2- to 5-ml syringe at a flow rate between 1 and 10 ml/min. Discard the liquid
eluted from the column. If the sample volume is less than 1.5 ml, change to buffer and proceed with the
injection until a total of 1.5 ml has been eluted. Discard the eluted liquid.
4. Elute the protein with the appropriate volume selected from Table 28. Collect the desalted protein in the
volume indicated.
The maximum recommended sample volume is 1.5 ml (when using one HiTrap Desalting 5-ml
column). See Table 28 for the effect of reducing the sample volume applied to the column.
Table 28. Recommended sample and elution volumes using a syringe, with examples of typical yields and remaining salt in the
desalted sample.
Sample load Add buffer Elute and collect Yield % Remaining Dilution factor
ml ml ml salt %
0.25 1.25 1.0 > 95 0.0 4.0
0.50 1.0 1.5 > 95 < 0.1 3.0
1.00 0.5 2.0 > 95 < 0.2 2.0
1.50 0.0 2.0 > 95 < 0.2 1.3
The void volume of the column is 1.5 ml. High molecular weight components elute between 1.5
and 4.5 ml, depending on the sample volume. Low molecular weight components start to elute
after 3.5 ml.
Note: Certain types of molecules, such as small heterocyclic or homocyclic aromatic compounds
(purines, pyrimidines, dye stuffs) can interact with Sephadex and are therefore eluted later than
expected. Larger sample volumes can be used in these cases, but the separation has to be optimized
for each case.
Desalting using a pump

1. Equilibrate the column; see Column equilibration.
2. Apply up to 1.5 ml of sample. Monitor the effluent from the column with a UV monitor and/or a conductivity
monitor. Keep the flow rate in the range 1 to 10 ml/min. Collect fractions.
3. Elute the column with approximately 10 ml of buffer before applying the next sample.
Collect fractions.

Desalting with ÄKTAprime plus
ÄKTAprime plus contains preprogrammed templates for individual HiTrap Desalting and HiPrep Desalting
26/10 columns. The procedure below uses a HiTrap Desalting 5-ml column.
Buffer preparation
Buffer (port A1): 20 mM sodium phosphate, 0.15 M NaCl, pH 7.0

Prepare at least 500 ml of the required buffer.
Sample preparation
Pass the sample through a 0.45 µm filter.
The maximum recommended sample volume is 1.5 ml.
Preparing ÄKTAprime plus

Once the system is prepared, the remaining steps (under Selecting Application Template and
starting the method) will be performed automatically.
1. Place the inlet tubing from port A (8-port valve) and port B (2-port valve) in the buffer.
5. Connect a sample loop large enough for your sample between ports 2 and 6 on the injection valve. Use a

2. Use the arrow and OK buttons to move in the menu tree until you find Desalting HiTrap Desalting.
Set Sample Inj. Vol
Templates
(00.0 ml) 00.0

Press OK to start
Desalting
Run data displayed
HiTrap Desalting
Figure 90 shows a theoretical desalting chromatogram, and Figure 91 shows typical results obtained
for buffer exchange of a histidine-tagged protein. The UV and conductivity traces enable the appropri-
ate desalted fractions to be pooled.

%B
100
Priming
50
& Equilibration
Elution
Sample
6 3 min
Fig 90. Theoretical gradient in Desalting, HiTrap Desalting Application Template. Total separation time = 9 min + sample application
time.
AU280 Sample: Histidine-tagged protein in 20 mM sodium phosphate,

0.5 M NaCl, 0.5 M imidazole, pH 7.4
UV 280 nm
Conductivity Column: HiTrap Desalting 5 ml
0.15 Buffer (A1): 20 mM sodium phosphate, 0.15 M NaCl, pH 7.0
Histidine-tagged
protein
0.10
0.05 Inject
0
0 1 min
Fig 91. Typical results for buffer exchange of a histidine-tagged protein.
Scaling up
For separation of sample volumes larger than 1.5 ml, or to increase the resolution between high and
low molecular weight components, up to five HiTrap Desalting columns can easily be connected in
series (see Table 27). For syringe operations, the volumes suggested in Table 28 should be increased
proportionally and the recommended flow rate maintained. The dilution of the sample is dependent on
the sample volume and the number of columns used in series. Lower dilution factors than those proposed
in Table 28 can be obtained, but the elution volumes have to be optimized for each combination of
sample volume and number of columns in series. The back pressure for each column is approximately
0.25 bar at 10 ml/min. For sample volumes up to 15 ml, HiPrep 26/10 Desalting is available (see below).
Up to four HiPrep 26/10 Desalting columns can be connected in series without passing the pressure
limit (up to 60 ml sample volume), assuming that the sample has essentially the same viscosity as the
eluent (see Table 27).

HiPrep 26/10 Desalting
Fig 92. Sixty-ml sample sizes can be run on four HiPrep 26/10 Desalting columns coupled in series.
HiPrep 26/10 Desalting is packed with Sephadex G-25 Fine. It provides group separation of high
(Mr > 5 000) from low molecular weight substances (Mr < 1 000), allowing reliable and reproducible
desalting and buffer exchange with sample sizes of 15 ml per column. Two to four columns can be
used in series for sample sizes of 30 to 60 ml. For more details, see Table 27.
Buffer exchange on HiPrep 26/10 Desalting with ÄKTAprime plus

Buffer preparation
Buffer (port A1): 20 mM sodium phosphate, 0.15 M NaCl, pH 7.0.

Sample preparation
Pass the sample through a 0.45 µm filter.
The maximum recommended sample volume is 15 ml.
Preparing ÄKTAprime plus

Once the system is prepared, the remaining steps (under Selecting Application Template and starting
the method) will be performed automatically.
1. Place the inlet tubing from port A (8-port valve) and port B (2-port valve) in the buffer.
5. Connect a sample loop large enough for your sample between port 2 and 6 on the injection valve.
Use a syringe to manually fill the loop.

2. Use the arrow and OK buttons to move in the menu tree until you find Desalting HiPrep Desalting.
Set Sample Inj. Vol
Templates
(00.0 ml) 00.0

Press OK to start
Desalting
Run data displayed
HiPrep Desalting
%B
100
Priming
50 & Equilibration
Elution
Sample
13 5 min
Fig 93. Theoretical gradient in Desalting, HiPrep Desalting Application Template. Total separation time = 18 min + sample application time.
AU280 Sample: BSA and sodium chloride

0.5 UV 280 nm Column: HiPrep 26/10 Desalting
BSA Conductivity Buffer (port A1): 20 mM phosphate, 0.15 M NaCl, pH 7.0
0.4
0.3
0.2
0.1 Inject
0 1 2 3 min
Fig 94. Typical results for buffer exchange of BSA.

Appendix 1
Characteristics of Ni Sepharose and uncharged
IMAC Sepharose products
Ni Sepharose products
Ni Sepharose High Performance is recommended for high-resolution purification of histidine-tagged
proteins, providing sharp peaks and concentrated eluate. Ni Sepharose 6 Fast Flow is excellent for
scaling up and batch purifications.
Table 29 summarizes key characteristics of bulk Ni Sepharose media, and Table 30 lists the stability of
the media under various conditions. Tables 31 to 37 summarize the characteristics of these same
media as prepacked columns and as prepacked 96-well plates. For more information, refer to Chapter 3.
Table 29. Characteristics of Ni Sepharose High Performance and Ni Sepharose 6 Fast Flow.
Characteristics Ni Sepharose High Performance Ni Sepharose 6 Fast Flow

Matrix Highly cross-linked 6% agarose, Highly cross-linked 6% agarose, precharged
precharged with Ni2+ with Ni2+
Metal ion capacity Approx. 15 µmol Ni2+/ml medium Approx. 15 µmol Ni2+/ml medium
Average particle size 34 µm 90 µm
Dynamic binding capacity1 At least 40 mg (histidine)6-tagged Approx. 40 mg (histidine)6-tagged protein/ml
protein/ml medium medium
Recommended flow rate2 < 150 cm/h 50–400 cm/h
Compatibility during use Stable in all commonly used buffers, Stable in all commonly used buffers,
reducing agents, denaturing agents reducing agents, denaturing agents and
and detergents. See Table 30 for detergents. See Table 30 for more
more information. information.
Chemical stability3 For one week at 40°C: 0.01 M HCl, For one week at 40°C: 0.01 M HCl,
0.1 M NaOH 0.1 M NaOH
For 12 h: 1 M NaOH, 70% acetic acid For 12 h: 1 M NaOH, 70% acetic acid
30 min tested: 30% 2-propanol 30 min tested: 30% 2-propanol
1 h tested: 2% SDS 1 h tested: 2% SDS
pH stability3 Short term (< 2 hours) 2–14 Short term (< 2 hours) 2–14
Long term (< 1 week) 3–12 Long term (< 1 week) 3–12
Storage 20% ethanol 20% ethanol
Storage temperature 4–30°C 4–30°C
1 Dynamic binding capacity conditions:
Sample: 1 mg/ml (histidine)6-tagged pure protein (Mr 43 000) in binding buffer or (histidine)6-tagged protein (Mr 28 000) bound
from E. coli extract. Capacity at 10% breakthrough.
Column volume: 0.25 ml or 1 ml
Flow rate: 0.25 ml/min or 1 ml/min, respectively
Binding buffer: 20 mM sodium phosphate, 0.5 M NaCl, 5 mM imidazole, pH 7.4
Elution buffer: 20 mM sodium phosphate, 0.5 M NaCl, 0.5 M imidazole, pH 7.4
Note: Dynamic binding capacity is protein dependent.
2 H2O at room temperature.

Table 30. Compatibility guide: Ni Sepharose High Performance and Ni Sepharose 6 Fast Flow are stable toward these compounds at
least at the concentrations given.
Compound Concentration
Reducing agents1 5 mM DTE
5 mM DTT
20 mM β-mercaptoethanol
5 mM TCEP
Denaturing agents 8 M urea2
6 M guanidine-HCl2
Detergents 2% Triton X-100 (nonionic)
2% Tween 20 (nonionic)
2% NP-40 (nonionic)
2% cholate (anionic)
1% CHAPS (zwitterionic)
Other additives 20% ethanol
50% glycerol
500 mM imidazole
100 mM Na2SO4
1.5 M NaCl
1 mM EDTA3
60 mM citrate2
Buffers 50 mM sodium phosphate, pH 7.4
100 mM Tris-HCl, pH 7.4
100 mM Tris-acetate, pH 7.4
100 mM HEPES, pH 7.4
100 mM MOPS, pH 7.4
100 mM sodium acetate, pH 42
1 Before performing runs with sample/buffers containing reducing reagents, a blank run with binding and elution buffers excluding
reducing agents is recommended, see page 33.

2 Tested for one week at 40°C.
3 The strong chelator EDTA has been used successfully in some cases, at 1 mM. Generally, chelating agents should be used with caution
(and only in the sample, not in the buffers). Any metal-ion stripping may be counteracted by addition of a small excess of MgCl2 before
centrifugation/filtration of the sample. Note that stripping effects may vary with applied sample volume.
Table 31. Characteristics of His MultiTrap HP and His MultiTrap FF.
Media His MultiTrap HP: Ni Sepharose High Performance

His MultiTrap FF: Ni Sepharose 6 Fast Flow
Filter plate size1 127.8 × 85.5 × 30.6 mm
Filter plate material Polypropylene and polyethylene
Binding capacity2 His MultiTrap HP: Up to 1 mg histidine-tagged protein/well
His MultiTrap FF: Up to 0.8 mg histidine-tagged protein/well
Reproducibility between wells +/- 10%
Volume packed medium/well 50 µl
Number of wells 96
Well volume 800 µl
Max. sample loading volume 600 µl
pH stability3 2–14 (short term), 3–12 (long term)
Storage 20% ethanol
Storage temperature 4–30°C
1According to ANSI/SBS 1-2004, 3-2004, and 4-2004 standards (ANSI = American National Standards and SBS = Society for
Biomolecular Screening).
2 Protein binding capacity is protein dependent.

Table 32. Characteristics of His SpinTrap.
Medium Ni Sepharose High Performance

Average particle size 34 µm
Bed volume 100 µl
Column material Polypropylene barrel and polyethylene frits
Protein binding capacity1 Approx. 0.75 mg histidine-tagged protein/column
Compatibility during use Stable in all commonly used buffers, reducing agents, denaturants and
detergents. See Table 30 for more information.
Storage 0.15% Kathon CG
Table 33. Characteristics of HisTrap HP and HisTrap FF.
Media HisTrap HP: Ni Sepharose High Performance

HisTrap FF: Ni Sepharose 6 Fast Flow
Column volume 1 ml and 5 ml
Column dimensions 0.7 × 2.5 cm (1 ml); 1.6 × 2.5 cm (5 ml)
Dynamic binding capacity1 HisTrap HP: At least 40 mg histidine-tagged protein/ml medium
HisTrap FF: Approx. 40 mg histidine-tagged protein/ml medium
Recommended flow rate 1 ml/min (1 ml); 5 ml/min (5 ml)
Max. flow rate2 4 ml/min (1 ml); 20 ml/min (5 ml)
Max. pressure2 0.3 MPa, 3 bar
Compatibility Stable in all commonly used buffers, reducing agents, denaturants and
Chemical stability3 For one week at 40°C: 0.01 M HCl, 0.1 M NaOH
For 12 h: 1 M NaOH, 70% acetic acid
30 min tested: 30% 2-propanol
1 h tested: 2% SDS
Storage 20% ethanol

Table 34. Characteristics of HisTrap FF crude.
Medium Ni Sepharose 6 Fast Flow

Column volume 1 ml and 5 ml
Dynamic binding capacity1 Approx. 40 mg histidine-tagged protein/ml medium
Recommended flow rate2 1 ml/min (1 ml); 5 ml/min (5 ml)
Max. pressure2 3 bar (0.3 MPa, 42 psi)
Compatibility during use Stable in all commonly used buffers, reducing agents, denaturing agents
and detergents. See Table 30 for more information.
1 h tested: 2% SDS
Storage 20% ethanol
Table 35. Characteristics and contents of HisTrap FF crude Kit.
Contents of kit 3 × 1 ml HisTrap FF crude columns 1

2 × 50 ml phosphate buffer, 8× stock, pH 7.4
50 ml 2 M imidazole, pH 7.4
1 syringe, 5 ml
Connectors
Instructions
1 See Table 34 for the characteristics of HisTrap FF crude columns.
Table 36. Characteristics of His GraviTrap.

Bed volume 1 ml
Column material Polypropylene barrel, polyethylene frits
Protein binding capacity1 Approx. 40 mg histidine-tagged protein/column
Compatibility during use Stable in all commonly used buffers, reducing agents, denaturing agents
and detergents. See Table 30 for more information.
1 h tested: 2% SDS
Storage 20% ethanol

Table 37. Characteristics of HisPrep FF 16/10.

Column volume 20 ml
Column dimensions 1.6 × 10 cm
Dynamic binding capacity1 Approx. 40 mg histidine-tagged protein/ml medium
Recommended flow rate2 2–10 ml/min (60–300 cm/h)
Max. flow rate2 10 ml/min (300 cm/h)
Max. pressure over the packed
bed during operation2 1.5 bar (0.15 MPa, 22 psi)
Column hardware
pressure limit 5 bar (0.5 MPa, 73 psi)
Compatibility during use Stable in all commonly used buffers, reducing agents, denaturing
agents and detergents. See Table 30 for more information.
1 h tested: 2% SDS
Storage 20% ethanol

Stripping, recharging, and cleaning of Ni Sepharose products
Stripping and recharging
Ni Sepharose High Performance and Ni Sepharose 6 Fast Flow do not have to be stripped and recharged
between each purification if the same protein is to be purified. It may be sufficient to strip and recharge it
after approximately two to five purifications, depending on the specific sample, sample pretreatment,
sample volume, etc.
Stripping buffer: 20 mM sodium phosphate, 500 mM NaCl, 50 mM EDTA, pH 7.4
1. Strip the media by washing with at least 5 to 10 column volumes of stripping buffer.
2. Wash with at least 5 to 10 column volumes of binding buffer.
3. Immediately wash with 5 to 10 column volumes of distilled water.
4. Recharge the water-washed column by loading 0.5 column volumes of 0.1 M NiSO4 in distilled water onto
the column.
5. Wash with 5 column volumes of distilled water, and 5 column volumes of binding buffer (to adjust pH)
before storage in 20% ethanol. Salts of other metals, chlorides, or sulfates may also be used.
It is important to wash with binding buffer as the last step to obtain the correct pH before
storage.
Cleaning-in-place
When an increase in back pressure is seen, the medium should be cleaned. Before cleaning,
strip off metal ions using the recommended procedure described above. The stripped medium
can be cleaned by the following methods:
To remove ionically bound protein:

1. Wash with several column volumes of 1.5 M NaCl.
2. Immediately wash with approximately 10 column volumes of distilled water.
To remove precipitated proteins, hydrophobically bound proteins, and lipoproteins:

1. Wash the column with 1 M NaOH, contact time usually 1 to 2 h (12 h or more for endotoxin removal).
2. Immediately wash with approximately 10 column volumes of binding buffer, followed by 5 to10 column
volumes of distilled water.
To remove hydrophobically bound proteins, lipoproteins, and lipids:

1. Wash with 5 to 10 column volumes of 30% isopropanol for about 15 to 20 min.
2a. Alternatively, wash with 2 column volumes of detergent in a basic or acidic solution. Use, for example,
0.1 to 0.5% nonionic detergent in 0.1 M acetic acid, contact time 1 to 2 h. After treatment , always remove
residual detergent by washing with at least 5 column volumes of 70% ethanol. Then wash with approxi-
mately 10 column volumes of distilled water.
Reversed flow may improve the efficiency of the cleaning-in-place procedure. After cleaning,
store in 20% ethanol (wash with 5 column volumes) or recharge with Ni2+ prior to storage in
ethanol.

Uncharged IMAC Sepharose products
IMAC Sepharose High Performance is recommended for high-resolution purifications, providing sharp
peaks and concentrated eluate. IMAC Sepharose 6 Fast Flow is excellent for scaling up.
Table 38 summarizes key characteristics of IMAC Sepharose media, and Table 39 lists the stability of
the media under various conditions. Tables 40 and 41 summarize the characteristics of the media as
prepacked columns. For more information, refer to Chapter 3.
Table 38. Characteristics of IMAC Sepharose High Performance and IMAC Sepharose 6 Fast Flow.
Characteristics IMAC Sepharose High Performance IMAC Sepharose 6 Fast Flow

Matrix Highly cross-linked 6% Highly cross-linked 6% spherical
spherical agarose agarose
Metal ion capacity Approx. 15 µmol Ni2+/ml medium Approx. 15 µmol Ni2+/ml medium
Average particle size 34 µm 90 µm
Dynamic binding capacity1 At least 40 mg (histidine)6-tagged Histidine-tagged protein:
protein/ml medium (Ni2+-charged) Approx. 40 mg (histidine)6-tagged
protein/ml medium (Ni2+-charged)
Untagged protein:
Approx. 25 mg/ml medium
(Cu2+-charged); approx. 15 mg/ml
medium (Zn2+- or Ni2+-charged).
Recommended flow rate2 < 150 cm/h 150 cm/h
Compatibility during use Stable in all commonly used buffers, Stable in all commonly used buffers,
reducing agents, denaturing agents reducing agents, denaturing agents
and detergents. See Table 39 for and detergents. See Table 39 for
more information. more information.
Chemical stability3 For one week at 40°C: 0.01 M HCl, For one week at 40°C: 0.01 M HCl,
0.1 M NaOH 0.1 M NaOH
For 12 h: 1 M NaOH, 70% acetic acid For 12 h: 1 M NaOH, 70% acetic acid
30 min tested: 30% 2-propanol 30 min tested: 30% 2-propanol
1 h tested: 2% SDS 1 h tested: 2% SDS
pH stability3 Short term (< 2 hours): 2–14 Short term (< 2 hours): 2–14
Long term (< 1 week): 3–12 Long term (< 1 week): 3–12
Storage 20% ethanol 20% ethanol
Storage temperature 4–30°C 4–30°C
1 Conditions for determining dynamic binding capacity:
Samples: (Histidine)6-tagged proteins: Capacity data were obtained for a protein (Mr 28 000) bound from an E. coli extract, and a
pure protein (Mr 43 000) applied at 1 mg/ml in binding buffer; capacity at 10% breakthrough.
Untagged protein (IMAC Sepharose 6 Fast Flow only): Capacities determined at 10% breakthrough for human
apotransferrin applied at 1 mg/ml in binding buffer.
Binding buffer: 20 mM sodium phosphate, 0.5 M NaCl, 5 mM imidazole (1 mM for untagged protein, IMAC Sepharose 6 Fast Flow only), pH 7.4
Elution buffer: 20 mM sodium phosphate, 0.5 M NaCl, 500 mM imidazole (50 mM for untagged protein, IMAC Sepharose 6 Fast Flow
only), pH 7.4
Note: Dynamic binding capacity is metal ion and protein dependent.
3 Uncharged medium only. See Table 39 for more information.

Table 39. Compatibility guide: IMAC Sepharose High Performance and IMAC Sepharose 6 Fast Flow are stable toward these
compounds at least at the concentrations given.
Compound Concentration
Reducing agents1 5 mM DTE
5 mM DTT
20 mM β-mercaptoethanol
5 mM TCEP
Denaturing agents 8 M urea2
6 M guanidine-HCl2
Detergents 2% Triton X-100 (nonionic)
2% Tween 20 (nonionic)
2% NP-40 (nonionic)
2% cholate (anionic)
1% CHAPS (zwitterionic)
Other additives 20% ethanol
50% glycerol
500 mM imidazole
100 mM Na2SO 4
1.5 M NaCl
1 mM EDTA3
60 mM citrate 2
Buffers 50 mM sodium phosphate, pH 7.4
100 mM Tris-HCl, pH 7.4
100 mM Tris-acetate, pH 7.4
100 mM HEPES, pH 7.4
100 mM MOPS, pH 7.4
100 mM sodium acetate, pH 4 2
1 Before performing runs with sample/buffers containing reducing reagents, a blank run with binding and elution buffers excluding
reducing agents is recommended (see page 79).

2 Tested for one week at 40°C.
3 The strong chelator EDTA has been used successfully in some cases at 1 mM. Generally, chelating agents should be used with caution
(and only in the sample, not in the buffers). Any metal-ion stripping may be counteracted by adding a small excess of MgCl2 before
centrifuging/filtrating the sample. Note that stripping effects may vary with applied sample volume.
Table 40. Characteristics of HiTrap IMAC HP and HiTrap IMAC FF.
Media HiTrap IMAC HP: IMAC Sepharose High Performance

HiTrap IMAC FF: IMAC Sepharose 6 Fast Flow
Column volume 1 ml or 5 ml
Dynamic binding capacity1 At least 40 mg histidine-tagged protein/ml medium when charged with Ni2+.
For untagged proteins, HiTrap FF can bind approx. 25 mg/ml medium
charged with Cu2+ or approx. 15 mg/ml medium charged with Zn2+ or Ni2+.
Recommended flow rate 1 ml/min (1 ml); 5 ml/min (5 ml)
Max flow rate2 4 ml/min (1 ml); 20 ml/min (5 ml)
Max. back pressure2 0.3 MPa, 3 bar

Table 40. Characteristics of HiTrap IMAC HP and HiTrap IMAC FF (continued).
1 h tested: 2% SDS
Storage 20% ethanol
pure protein (Mr 43 000) applied at 1 mg/ml in binding buffer; capacity at 10% breakthrough.
Untagged protein (IMAC Sepharose 6 Fast Flow only): Capacities determined at 10% breakthrough for human
apotransferrin applied at 1 mg/ml in binding buffer.
Binding buffer: 20 mM sodium phosphate, 0.5 M NaCl, 5 mM imidazole (1 mM for untagged protein, IMAC Sepharose 6 Fast Flow only), pH 7.4
Elution buffer: 20 mM sodium phosphate, 0.5 M NaCl, 500 mM imidazole (50 mM for untagged protein, IMAC Sepharose 6 Fast Flow
only), pH 7.4.
Table 41. Characteristics of HiPrep IMAC FF 16/10.
Medium IMAC Sepharose 6 Fast Flow

Column volume 20 ml
Dynamic binding capacity1 Approx. 40 mg histidine-tagged protein/ml medium when charged with Ni2+.
For untagged proteins, HiTrap FF binds approx. 25 mg/ml medium charged
with Cu2+ or approx. 15 mg/ml medium charged with Zn2+ or Ni2+.
bed during operation2 0.15 MPa, 1.5 bar
Column hardware pressure limit 0.5 MPa, 5 bar
1 h tested: 2% SDS
Storage 20% ethanol
pure protein (Mr 43 000) applied at 1 mg/ml in binding buffer; capacity at 10 % breakthrough.
Untagged protein: Capacities determined at 10 % breakthrough for human apo-transferrin applied at 1 mg/ml in
binding buffer.
Column volume: 0.25 or 1 ml
Flow rate: 0.25 or 1 ml/min, respectively
Binding buffer: 20 mM sodium phosphate, 500 mM NaCl, 5 mM imidazole, (1 mM for untagged protein) pH 7.4
Elution buffer: 20 mM sodium phosphate, 500 mM NaCl, 500 mM imidazole, (50 mM for untagged protein) pH 7.4

Stripping, recharging, and cleaning of IMAC Sepharose products
IMAC Sepharose High Performance and IMAC Sepharose 6 Fast Flow do not have to be stripped and
recharged between each purification if the same protein is to be purified. It may be sufficient to strip
and recharge medium after approximately two to five purifications, depending on the specific sample,
sample pretreatment, sample volume, etc.
Stripping and recharging

Stripping buffer: 20 mM sodium phosphate, 500 mM NaCl, 50 mM EDTA, pH 7.4
1. Strip the medium by washing with at least 5 to 10 column volumes of stripping buffer.
2. Wash with at least 5 to 10 column volumes of binding buffer.
3. Immediately wash with 5 to 10 column volumes of distilled water.
4. Prepare a 0.1 M solution of the chosen metal ion in distilled water. Salts of chlorides, sulfates, etc., can be
used: e.g., 0.1 M CuSO 4 or 0.1 M NiSO4.
5. Recharge the water-washed column by loading at least 0.5 column volume of 0.1 M metal ion/salt solution.
6. Wash with 5 column volumes of distilled water, and 5 column volumes of binding buffer (to adjust pH)
before storing column in 20% ethanol.
It is important to wash with binding buffer as the last step to obtain the correct pH before
storage.
Cleaning-in-place
When an increase in back pressure is seen, the medium should be cleaned. Before cleaning,
strip off metal ions using the recommended procedure described above. The stripped medium
can be cleaned by the following methods:
To remove ionically bound protein:

1. Wash with several column volumes of 1.5 to 2.0 M NaCl.
2. Immediately wash with approximately 3 to 10 column volumes of distilled water.
To remove precipitated proteins, hydrophobically bound proteins, and lipoproteins:

1. Wash the column with 1 M NaOH, contact time usually 1 to 2 h (12 h or more for endotoxin removal).
2. Immediately wash with approximately 10 column volumes binding buffer, followed by 5 to 10 column
volumes distilled water.
To remove hydrophobically bound proteins, lipoproteins, and lipids:

1. Wash with 5 to 10 column volumes of 30% isopropanol for about 15 to 20 min.
2a. Alternatively, wash with 2 column volumes of detergent in a basic or acidic solution. Use, for example,
0.1 to 0.5% nonionic detergent in 0.1 M acetic acid, contact time 1 to 2 h. After treatment , always remove
residual detergent by washing with at least 5 column volumes of 70% ethanol. Then wash with approxi-
mately 10 column volumes of distilled water.
Reversed flow may improve the efficiency of the cleaning-in-place procedure. After cleaning,
store column in 20% ethanol (wash with 5 column volumes) or recharge with metal ions prior to
storing in ethanol.

Appendix 2
Characteristics of Glutathione Sepharose
products
Glutathione Sepharose High Performance is recommended for high-resolution purification of GST-
tagged proteins, providing sharp peaks and concentrated eluent. Glutathione Sepharose Fast Flow is
excellent for scaling up. Glutathione Sepharose 4B is recommended for packing small columns and
other formats including batch purifications.
Table 42 summarizes key characteristics of these three Glutathione Sepharose media, and Table 43
lists the stability of the media toward various compounds under various conditions. Tables 44 to 45
summarize the characteristics of the same media prepacked as GSTrap HP, GSTrap FF, and GSTrap 4B
in columns and as 96-well plates. For more information, refer to Chapter 5.
Table 42. Characteristics of Glutathione Sepharose High Performance, Glutathione Sepharose 4 Fast Flow, and Glutathione Sepharose 4B.
Characteristics Glutathione Sepharose Glutathione Glutathione

High Performance Sepharose 4 Fast Flow Sepharose 4B
Matrix Highly cross-linked Highly cross-linked 4% agarose
6% agarose 4% agarose
Average 34 µm 90 µm 90 µm
particle size
Ligand 1.5–3.5 mg glutathione/ml 120–320 µmol glutathione/ml 200–400 µmol glutathione/g
concentration medium (based on Gly) medium washed and dried medium
Binding > 10 mg recombinant > 10 mg recombinant > 5 mg recombinant
capacity1 glutathione glutathione glutathione
S-transferase/ml medium S-transferase/ml medium S-transferase/ml medium
Recommended < 150 cm/h 50–300 cm/h < 75 cm/h
flow rate2
Chemical Stable to all commonly Stable to all commonly used Stable to all commonly used
stability used aqueous buffers, e.g. aqueous buffers, e.g. aqueous buffers. Exposure to
1 M acetate, pH 4.0 and 1 M acetate, pH 4.0, and 0.1 M NaOH, 70% ethanol, or
6 M guanidine hydrochloride 6 M guanidine hydrochloride 6 M guanidine hydrochloride
for 1 h at room temperature for 1 h at room temperature for 2 h at room temperature
or to 1% (w/v) SDS for 14 d
causes no significant loss
of activity.
pH stability 3–12 3–12 4–13
Storage 4–30°C 4–30°C 4–8°C
temperature
Storage buffer 20% ethanol 20% ethanol 20% ethanol
1 Thebinding of GST-tagged proteins depends on size, conformation, and concentration of the protein in the sample loaded. Binding of
GST to glutathione is also flow dependent, and lower flow rates often increase the binding capacity. This is important during sample
loading. Protein characteristics, pH, and temperature, but also the media used may affect the binding capacity.

Table 43. Characteristics of GST MultiTrap FF and GST MultiTrap 4B.
Media GST MultiTrap FF: Glutathione Sepharose 4 Fast Flow

GST MultiTrap 4B: Glutathione Sepharose 4B
Filter plate size1 127.8 × 85.5 × 30.6 mm
Filter plate material Polypropylene and polyethylene
Binding capacity GST MultiTrap FF: Up to 0.5 mg GST-tagged protein/well
GST MultiTrap 4B: Up to 0.5 mg GST-tagged protein/well
Reproducibility between wells2 +/- 10%
Volume packed medium/well 50 µl (500 µl of 10% slurry)
Number of wells 96
Centrifugation speed: Depends on sample pretreatment and sample properties
recommended 100–500 × g
maximum 700 × g
Vacuum pressure: Depends on sample pretreatment and sample properties
recommended -0.1 to -0.3 bar
maximum -0.5 bar
pH stability Glutathione Sepharose 4 Fast Flow: 3–12
Glutathione Sepharose 4B: 4–13
Storage 20% ethanol
1According to ANSI/SBS 1-2004, 3-2004, and 4-2004 standards (ANSI = American National Standards and SBS = Society for
Biomolecular Screening).
2 The amount of eluted target proteins/well does not differ more than +/- 10% from the average amount/well for the entire filter plate.
Table 44. Characteristics of prepacked GSTrap HP, GSTrap HP, and GSTrap 4B columns.
Characteristics GSTrap HP GSTrap FF GSTrap 4B

Media Glutathione Sepharose Glutathione Sepharose 4 Glutathione Sepharose 4B
High Performance Fast Flow
Average 34 µm 90 µm 90 µm
particle size
Dynamic binding Approx. 10 mg GST-tagged Approx. 11 mg GST-tagged Approx. 10 mg recombinant
capacity1,2 protein/ml medium, protein/ml medium, glutathione S-transferase
Mr 63 000 Mr 43 000 (Mr 26 000)/ml medium
Max. back 0.3 MPa, 3 bar 0.3 MPa, 3 bar 0.3 MPa, 3 bar
pressure3
Recommended Sample loading: Sample loading: Sample loading:
flow rate3 0.2–1 ml/min (1 ml ) and 0.2–1 ml/min (1 ml ) and 0.2–1 ml/min (1 ml) and
1–5 ml (5 ml) 1–5 ml (5 ml) 0.5–5 ml/min (5 ml)
Washing and elution: Washing and elution: Washing and elution:
1–2 ml/min (1 ml) and 1–2 ml/min (1 ml) and 1 ml/min (1 ml) and
5–10 ml/min (5 ml) 5–10 ml/min (5 ml) 5 ml/min (5 ml)
Chemical Stable to all commonly used Stable to all commonly used Stable to all commonly used
stability aqueous buffers, e.g. aqueous buffers, e.g. aqueous buffers. Exposure to
1 M acetate, pH 4.0 and 1 M acetate, pH 6.0, and 0.1 M NaOH, 70% ethanol, or
6 M guanidine hydrochloride 6 M guanidine hydrochloride 6 M guanidine hydrochloride
for 1 h at room temperature for 1 h at room temperature for 2 h at room temperature
or to 1% (w/v) SDS for 14 d
causes no significant loss
of activity.

Table 44. Characteristics of prepacked GSTrap HP, GSTrap HP, and GSTrap 4B columns (continued).
Characteristics GSTrap HP GSTrap FF GSTrap 4B

pH stability 3–12 3–12 4–13
Storage 4–30°C 4–30°C 4–8°C
temperature
Storage buffer 20% ethanol 20% ethanol 20% ethanol
The column dimensions are identical for all three GSTrap columns (0.7 × 2.5 cm for the 1-ml column and 1.6 × 2.5 cm for the 5-ml column).
Column volumes are 1 ml and 5 ml.
1 Thebinding of GST-tagged proteins depends on size, conformation, and concentration of the protein in the sample loaded. Binding of
2 Dynamic binding capacity conditions (60% breakthrough):
Sample: 1 mg/ml pure GST-tagged protein in binding buffer
Column volume: 0.4 ml
Flow rate: 0.2 ml/min (60 cm/h)
Binding buffer: 10 mM sodium phosphate, 140 mM NaCl, 2.7 mM KCl, pH 7.4
Table 45. Characteristics of GSTPrep FF 16/10.
Characteristics GSTPrep FF 16/10

Medium Glutathione Sepharose 4 Fast Flow
Column volume 20 ml
Dynamic binding capacity1,2 Approx. 11 mg GST-tagged protein/ml medium, Mr 43 000
bed during operation3 1.5 bar (0.15 MPa, 22 psi)
Column hardware pressure limit 5 bar (0.5 MPa, 73 psi)
Storage 20% ethanol
1 The binding of GST-tagged proteins depends on size, conformation, and concentration of the protein in the sample loaded. Binding of
2 Dynamic binding capacity conditions (60% breakthrough):
Sample: 1 mg/ml pure GST-tagged protein in binding buffer
Column volume: 0.4 ml
Flow rate: 0.2 ml/min (60 cm/h)
Binding buffer: 10 mM sodium phosphate, 140 mM NaCl, 2.7 mM KCl, pH 7.4

Cleaning of Glutathione Sepharose products
The procedure below is appropriate for use with both bulk media and prepacked columns.
Reuse of purification columns and media depends upon the nature of the sample and should
only be performed with identical samples to prevent cross contamination.
If required, Glutathione Sepharose High Performance, Glutathione Sepharose 4 Fast Flow, and
Glutathione Sepharose 4B media and prepacked columns can be regenerated for reuse as
follows:
1. Wash with 2 to 3 column volumes of alternating high pH (0.1 M Tris-HCl, 0.5 M NaCl, pH 8.5) and low pH
(0.1 M sodium acetate, 0.5 M NaCl, pH 4.5) buffers.
2. Repeat the cycle 3 times.
3. Reequilibrate with 3 to 5 column volumes of PBS, pH 7.3.
If Glutathione Sepharose appears to be losing binding capacity, it may be due to an accumulation of

precipitated, denatured, or nonspecifically bound proteins.
To remove precipitated or denatured substances:

1. Wash with 2 column volumes of 6 M guanidine hydrochloride.
2. Immediately wash with 5 column volumes of PBS, pH 7.3.
To remove hydrophobically bound substances:

1. Wash with 3 to 4 column volumes of 70% ethanol (or 2 column volumes of 1% Triton X-100).
2. Immediately wash with 5 column volumes of PBS, pH 7.3.
For long-term storage (> 1 month):

1. Wash the column twice with 5 to 10 column volumes of PBS, pH 7.3.
2. Repeat washes using 20% ethanol.
3. Store at 4 to 8°C.
4. Reequilibrate the column with 5 to 10 column volumes of PBS, pH 7.3 before reuse.

Appendix 3
Precipitation and resolubilization
Specific sample preparation steps may be required if the crude sample is known to contain contaminants
such as lipids, lipoproteins, or phenol red that may build up on a column or if certain gross impurities,
such as bulk protein, should be removed before any chromatographic step.
Fractional precipitation
Fractional precipitation is occasionally used at laboratory scale to remove gross impurities from small
sample volumes and also in small-scale commercial production. When using a HiTrap affinity purification
column, for example, a HisTrap or GSTrap column, at laboratory scale, it is unlikely that fractional
precipitation will be required.
Precipitation techniques separate fractions by the principle of differential solubility. For example, because
protein species differ in their degree of hydrophobicity, increased salt concentrations can enhance
hydrophobic interactions between the proteins and cause precipitation. Fractional precipitation can be
applied to remove gross impurities in three different ways, as shown in Figure 95.
Clarification Supernatant
Bulk proteins and
particulate matter
precipitated
Extraction, Clarification,
Concentration Redissolve Purification
Target protein precipitated pellet* Remember: if precipitating agent is
with proteins of similar incompatible with next purification
solubility step, use Sephadex G-25 for desalting
Concentration and buffer exchange, e.g., HiTrap Desalting,
PD-10 columns, or HiPrep 26/10
Extraction, Clarification Target protein
Redissolve Desalting column
Bulk proteins and precipitated
particulate matter with proteins pellet*
precipitated of similar *Remember: not all proteins are easy
to redissolve, yield may be reduced
solubility
Fig 95. Three ways to use precipitation.
Precipitation techniques may be affected by temperature, pH, and sample concentration. These
parameters must be controlled to ensure reproducible results.
Most precipitation techniques are not suitable for large-scale preparation.
Examples of precipitation agents are reviewed in Table 46. The most common precipitation method
using ammonium sulfate is described in more detail.

Table 46. Examples of precipitation techniques.
Precipitation agent Typical conditions Sample type Comment

for use
Ammonium sulfate As described below. > 1 mg/ml proteins Stabilizes proteins, no
especially immuno- denaturation; supernatant
globulins. can go directly to HIC.
Helps to reduce lipid content .
Dextran sulfate Add 0.04 ml of 10% Samples with high Precipitates lipoprotein.
dextran sulfate and levels of lipoprotein,
1 ml of 1 M CaCl2 per ml e.g., ascites.
of sample, mix 15 min,
centrifuge at 10 000 × g,
discard pellet.
Polyvinylpyrrolidine Add 3% (w/v), stir 4 h, Samples with high Alternative to dextran sulfate.
centrifuge at 17 000 × g, levels of lipoprotein,
discard pellet. e.g., ascites.
Polyethylene glycol Up to 20% (w/v) Plasma proteins. No denaturation,
(PEG, Mr > 4000) supernatant goes directly
to IEX or AC, complete
removal may be difficult.
Stabilizes proteins.
Acetone (cold) Up to 80% (v/v) May denature protein
at 0°C. Collect pellet irreversibly.
after centrifugation Useful for peptide precipitation
at full speed in an or concentration of sample for
Eppendorf centrifuge. electrophoresis.
Polyethyleneimine 0.1% (w/v) Precipitates aggregated
nucleoproteins.
Protamine sulfate 1% (w/v) Precipitates aggregated
nucleoproteins.
Streptomycin sulfate 1% (w/v) Precipitates nucleic acids.
Caprylic acid (X/15) g where Antibody concentration Precipitates bulk of proteins
X = volume of sample. should be > 1 mg/ml. from sera or ascites, leaving
immunoglobulins in solution.
Details taken from: Scopes R.K., Protein Purification, Principles and Practice, Springer, (1994), J.C. Janson and L. Rydén, Protein Purification,
Principles, High Resolution Methods and Applications, 2nd ed. Wiley Inc, (1998).
Ammonium sulfate precipitation

Ammonium sulfate precipitation is frequently used for initial sample concentration and clean-up. As
the concentration of the salt is increased, proteins will begin to “salt out.” Different proteins salt out at
different concentrations, a process that can be taken advantage of to remove contaminating proteins
from the crude extract. The salt concentration needs to be optimized to remove contaminants and not
the desired protein. An additional step with increased salt concentration should then precipitate the
target protein. If the target protein cannot be safely precipitated and redissolved, only the first step
should be employed. HIC is often an excellent followup, as the sample already contains a high salt
concentration and can be applied directly to the HIC column with little or no additional preparation.
The elevated salt level enhances the interaction between the hydrophobic components of the sample
and the chromatography medium.
Solutions needed for precipitation:
Saturated ammonium sulfate solution (add 100 g ammonium sulfate to 100 ml distilled water, stir to
dissolve).
1 M Tris-HCl, pH 8.0.
Buffer for first purification step.

Some proteins may be damaged by ammonium sulfate. Take care when adding crystalline
ammonium sulfate: high local concentrations may cause contamination of the precipitate with
unwanted proteins.
It may be practical to use HIC as second step after an initial ammonium sulfate precipitation.
For routine, reproducible purification, precipitation with ammonium sulfate should be avoided in
favor of chromatography.
In general, precipitation is rarely effective for protein concentrations below 1 mg/ml.
1. Filter (0.45 µm) or centrifuge the sample (10 000 × g at 4°C).

2. Add 1 part 1 M Tris-HCl, pH 8.0 to 10 parts sample volume to maintain pH.
3. Stir gently. Add ammonium sulfate solution, drop by drop. Add up to 50% saturation1.
Stir for 1 h.
4. Centrifuge 20 min at 10 000 × g.
5. Remove supernatant. Wash the pellet twice by resuspension in an equal volume of ammonium sulfate
solution of the same concentration (i.e., a solution that will not redissolve the precipitated protein or
cause further precipitation). Centrifuge again.
6. Dissolve pellet in a small volume of the buffer to be used for the next step.
7. Ammonium sulfate is removed during clarification/buffer exchange steps with Sephadex G-25, using
desalting columns (see Chapter 9).
1
The % saturation can be adjusted either to precipitate a target molecule or to precipitate contaminants.
The quantity of ammonium sulfate required to reach a given degree of saturation varies according to
temperature. Table 47 shows the quantities required at 20°C.
Table 47. Quantities of ammonium sulfate required to reach given degrees of saturation at 20°C.
Final percent saturation to be obtained

20 25 30 35 40 45 50 55 60 65 70 75 80 85 90 95 100
Starting Amount of ammonium sulfate to add (grams) per liter of solution at 20°C
percent
saturation
0 113 144 176 208 242 277 314 351 390 430 472 516 561 608 657 708 761
5 85 115 146 179 212 246 282 319 358 397 439 481 526 572 621 671 723
10 57 86 117 149 182 216 251 287 325 364 405 447 491 537 584 634 685
15 28 58 88 119 151 185 219 255 293 331 371 413 456 501 548 596 647
20 0 29 59 89 121 154 188 223 260 298 337 378 421 465 511 559 609
25 0 29 60 91 123 157 191 228 265 304 344 386 429 475 522 571
30 0 30 61 92 125 160 195 232 270 309 351 393 438 485 533
35 0 30 62 94 128 163 199 236 275 316 358 402 447 495
40 0 31 63 96 130 166 202 241 281 322 365 410 457
45 0 31 64 98 132 169 206 245 286 329 373 419
50 0 32 65 99 135 172 210 250 292 335 381
55 0 33 66 101 138 175 215 256 298 343
60 0 33 67 103 140 179 219 261 305
65 0 34 69 105 143 183 224 267
70 0 34 70 107 146 186 228
75 0 35 72 110 149 190
80 0 36 73 112 152
85 0 37 75 114
90 0 37 76
95 0 38

Appendix 4
Column packing and preparation
Prepacked columns from GE Healthcare will ensure reproducible results and the highest performance.
Use small prepacked columns or 96-well filter plates for media screening and method optimization
to increase efficiency in method development.
Efficient column packing is essential for a good separation, especially when using gradient elution.
A poorly packed column gives rise to poor and uneven flow, peak broadening, and loss of resolution.
If column packing is required, the following guidelines will apply at all scales of operation:
• When using a binding technique, use short, wide columns (typically 5 to 15 cm bed height) for rapid
purification, even with low linear flow.
• The amount of medium required will depend on the binding capacity of the medium and the amount
of sample. The binding capacity of a medium is always significantly influenced by the hydrophobic
nature of the sample as well as the medium itself and must be determined empirically. Estimate the
amount of medium required to bind the sample of interest and use five times this amount to pack the
column. The amount of medium required can be reduced if resolution is satisfactory.
• Once separation parameters have been determined, scale up a purification by increasing the diameter
of the column to increase column volume. Avoid increasing the length of the column as this will alter
separation conditions.
Affinity media for tagged proteins can be packed in either Tricorn or XK columns available from
GE Healthcare. A step-by-step demonstration of column packing can be seen in “Column Packing —
The Movie”, available in CD format (see Ordering information).
Fig 96. “Column Packing — The Movie” provides a step-by-step demonstration of column packing.

1. Equilibrate all materials to the temperature at which the separation will be performed.
2. Eliminate air by flushing column end pieces with the recommended buffer. Ensure no air is
trapped under the column net. Close column outlet leaving 1–2 cm of buffer in the column.
3. Gently resuspend the medium.
Note that affinity media from GE Healthcare are supplied ready to use. Decanting of fines that could
clog the column is unnecessary.
Avoid using magnetic stirrers because they may damage the matrix.
4. Estimate the amount of slurry (resuspended medium) required on the basis of the recommen-
dations supplied.
5. Pour the required volume of slurry into the column. Pouring down a glass rod held against the
wall of the column will minimize the introduction of air bubbles.
6. Immediately fill the column with buffer.
7. Mount the column top piece and connect to a pump.
8. Open the column outlet and set the pump to the desired flow rate (for example, 15 ml/min in
an XK 16/20 column).
When slurry volume is greater than the total volume of the column, connect a second glass
column to act as a reservoir (see Ordering information for details). This ensures that the slurry
has a constant diameter during packing, minimizing turbulence and improving column packing
conditions.
If the recommended flow rate cannot be obtained, use the maximum flow rate the pump can
deliver.
Do not exceed the maximum operating pressure of the medium or column.
9. Maintain the packing flow rate for at least 3 column volumes after a constant bed height is
obtained. Mark the bed height on the column.
Do not exceed 75% of the packing flow rate during any purification.
10. Stop the pump and close the column outlet. Remove the top piece and carefully fill the rest of
the column with buffer to form an upward meniscus at the top.
11. Insert the adaptor into the column at an angle, ensuring that no air is trapped under the net.
12. Slide the adaptor slowly down the column (the outlet of the adaptor should be open) until the
mark is reached. Lock the adaptor in position.
13. Connect the column to the pump and begin equilibration. Reposition the adaptor if necessary.
The medium must be thoroughly washed to remove the storage solution, usually 20% ethanol.
Residual ethanol may interfere with subsequent procedures.
Many media equilibrated with sterile phosphate-buffered saline containing an antimicrobial

agent may be stored at 4°C for up to 1 month, but always follow the specific storage instructions
supplied with the product.

Column selection
Tricorn and XK columns are fully compatible with the high flow rates achievable with modern media,
and a broad range of column dimensions are available (see Table 48). In most cases the binding
capacity of the medium and the amount of sample to be purified will determine the column size required.
Also, Empty Disposable PD-10 Columns are available for single-use applications using gravity flow. For
a complete listing of available columns, refer to the GE Healthcare catalog, BioDirectory, or
www.gehealthcare.com/protein-purification
Table 48. Column bed volumes and heights1.
Column Size
i.d. Length Bed Volume Bed Height
(mm) (ml) (cm)
Tricorn 5/20 5 20 mm 0.31–0.55 1.6–2.8
Tricorn 5/50 5 50 mm 0.90–1.14 4.6–5.8
Tricorn 10/20 10 20 mm 1.26–2.20 1.6–2.8
Tricorn 10/50 10 50 mm 3.61–4.56 4.6–5.8
Tricorn 10/100 10 100 mm 7.54–8.48 9.6–10.8
XK 16/20 16 20 cm 5–31 2.5–15
XK 16/40 16 40 cm 45–70 22.5–35
XK 26/20 26 18 cm 5.3–66 1–12.5
XK 26/40 26 40 cm 122–186 23–35
XK 50/20 50 18 cm 0–274 0–14
XK 50/30 50 30 cm 265–559 13.5–28.5
Empty Disposable PD-102 15 7.4 cm 8.3 4.8–5
1 All Tricorn and XK column specifications apply when one adapter is used.
2 Forgravity-flow applications. Together with LabMate Buffer Reservoir, up to 25 ml of buffer and/or sample can be applied, which
reduces handling time considerably.

Appendix 5
Conversion data: proteins, column pressures
Mass (g/mol) 1 µg 1 nmol
10 000 100 pmol; 6 x 1013 molecules 10 µg
50 000 20 pmol; 1.2 x 1013 molecules 50 µg
100 000 10 pmol; 6.0 x 1012 molecules 100 µg
150 000 6.7 pmol; 4.0 x 1012 molecules 150 µg
Protein A280 for 1 mg/ml

IgG 1.35
IgM 1.20
IgA 1.30
Protein A 0.17
Avidin 1.50
Streptavidin 3.40
Bovine Serum Albumin 0.70
1 kb of DNA = 333 amino acids of coding capacity

= 37 000 g/mol
270 bp DNA = 10 000 g/mol
1.35 kb DNA = 50 000 g/mol
2.70 kb DNA = 100 000 g/mol
Average molecular weight of an amino acid = 120 g/mol.
Column pressures
The maximum operating back pressure refers to the pressure above which the column contents may
begin to compress.
Pressure units may be expressed in megaPascals, bar or pounds per square inch and can be converted
as follows: 1 MPa = 10 bar = 145 psi

Appendix 6
Converting from linear flow (cm/h) to volumetric
flow rates (ml/min) and vice versa
It is convenient when comparing results for columns of different sizes to express flow as linear flow
(cm/h). However, flow is usually measured in volumetric flow rate (ml/min). To convert between linear
flow and volumetric flow rate use one of the formulas below.
From linear flow (cm/h) to volumetric flow rate (ml/min)

Volumetric flow rate (ml/min) = Linear flow (cm/h) x column cross sectional area (cm2)
60
2
= Y x π x d
60 4
where
Y = linear flow in cm/h
d = column inner diameter in cm
Example:
What is the volumetric flow rate in an XK 16/70 column (i.d. 1.6 cm) when the linear flow is 150 cm/h?
Y = linear flow = 150 cm/h
d = inner diameter of the column = 1.6 cm
Volumetric flow rate = 150 x π x 1.6 x 1.6 ml/min

60 x 4
= 5.03 ml/min
From volumetric flow rate (ml/min) to linear flow (cm/hour)

Linear flow (cm/h) = Volumetric flow rate (ml/min) x 60
column cross sectional area (cm2)
= Z x 60 x 4
π x d2
where
Z = volumetric flow rate in ml/min
d = column inner diameter in cm
Example:
What is the linear flow in a Tricorn 5/50 column (i.d. 0.5 cm) when the volumetric flow rate is 1 ml/min?
Z = Volumetric flow rate = 1 ml/min
d = column inner diameter = 0.5 cm
Linear flow = 1 x 60 x 4 cm/h

π x 0.5 x 0.5
= 305.6 cm/h
From ml/min to using a syringe

1 ml/min = approximately 30 drops/min on a HiTrap 1 ml column
5 ml/min = approximately 120 drops/min on a HiTrap 5 ml column

Appendix 7
GST vectors
Fig 97. Map of the GST vectors showing the reading frames and main features.

SELECTION GUIDE – pGEX Vector Control Regions
pGEX-2TK pGEX-4T-1 pGEX-4T-2 pGEX-4T-3 pGEX-5X-1 pGEX-5X-2 pGEX-5X-3 pGEX-6P-1 pGEX-6P-2 pGEX-6P-3
27-4587-01 27-4580-01 27-4581-01 27-4583-01 27-4584-01 27-4585-01 27-4586-01 27-4597-01 27-4598-01 27-4599-01
Glutathione S- Transferase Region
tac promoter
–10 205–211 205–211 205–211 205–211 205–211 205–211 205–211 205–211 205–211 205–211
–35 183–188 183–188 183–188 183–188 183–188 183–188 183–188 183–188 183–188 183–188
lac operator 217–237 217–237 217–237 217–237 217–237 217–237 217–237 217–237 217–237 217–237
Ribosome binding site for GST 244 244 244 244 244 244 244 244 244 244
Start codon (ATG) for GST 258 258 258 258 258 258 258 258 258 258
Coding region for
thrombin cleavage 918–935 918–935 918–935 918–935 NA NA NA NA NA NA
Coding region for
Factor Xa cleavage NA NA NA NA 921–932 921–932 921–932 NA NA NA
Coding region for PreScission
Protease cleavage NA NA NA NA NA NA NA 918–938 918–938 918–938
Coding for kinase
recognition site 936–950 NA NA NA NA NA NA NA NA NA
Multiple Cloning Site 951–966 930–966 930–967 930–965 934–969 934–970 934–971 945–981 945–982 945–980
r
β-lactamase (Amp ) Gene Region
Promoter
Control regions for pGEX vectors
–10 1330–1335 1330–1335 1331–1336 1329–1334 1333–1338 1334–1339 1335–1340 1345–1350 1346–1351 1344–1349
–35 1307–1312 1307–1312 1308–1313 1306–1311 1310–1315 1311–1316 1312–1317 1322–1327 1323–1328 1321–1326
Start codon (ATG) 1377 1377 1378 1376 1380 1381 1382 1392 1393 1391
Stop codon (TAA) 2235 2235 2236 2234 2238 2239 2240 2250 2251 2249
q
LacI Gene Region
Start codon (GTG) 3318 3318 3319 3317 3321 3322 3323 3333 3334 3332
Stop codon (TGA) 4398 4398 4399 4397 4401 4402 4403 4413 4414 4412
Plasmid Replication Region
Site of replication initiation 2995 2995 2996 2994 2998 2999 3000 3010 3011 3009
Region necessary
for replication 2302–2998 2302–2998 2303–2999 2301–2997 2305–3001 2306–3002 2307–3003 2317–3013 2318–3014 3216–3012
Sequencing Primers
5' pGEX Sequencing
Primer binding 869–891 869–891 869–891 869–891 869–891 869–891 869–891 869–891 869–891 869–891
3' pGEX Sequencing
Primer binding 1041–1019 1041–1019 1042–1020 1040–1018 1044–1022 1045–1023 1046–1024 1056–1034 1057–1035 1055–1033
GenBank Accession Number U13851 U13853 U13854 U13855 U13856 U13857 U13858 U78872 U78873 U78874
Complete DNA sequences and restriction site data are available with each individual vector's product information, at the GE Healthcare Web site (www.gehealthcare.com/lifesciences).

Appendix 8
Amino acids table
Three-letter Single-letter
Amino acid code code Structure
HOOC
CH3
Alanine Ala A
H2N
HOOC NH2
CH2CH2CH2NHC
Arginine Arg R
H2N NH
HOOC
CH2CONH2
Asparagine Asn N
H2N
HOOC
CH2COOH
Aspartic Acid Asp D
H2N
HOOC
CH2SH
Cysteine Cys C
H2N
HOOC
CH2CH2COOH
Glutamic Acid Glu E
H2N
HOOC
CH2CH2CONH2
Glutamine Gln Q
H2N
HOOC
H
Glycine Gly G
H2N
HOOC N
CH2
Histidine His H NH
H2N
HOOC
CH(CH3)CH2CH3
Isoleucine Ile I
H2N
HOOC CH3
CH2CH
Leucine Leu L
H2N CH3
HOOC
CH2CH2CH2CH2NH2
Lysine Lys K
H2N
HOOC
CH2CH2SCH3
Methionine Met M
H2N
HOOC
CH2
Phenylalanine Phe F
H2N
HOOC
Proline Pro P
NH
HOOC
CH2OH
Serine Ser S
H2N
HOOC
CHCH3
Threonine Thr T
H2N OH
HOOC
CH2
Tryptophan Trp W
H2N
NH
HOOC
CH2 OH
Tyrosine Tyr Y
H2N
HOOC
CH(CH3)2
Valine Val V
H2N

Middle unit
residue (-H20) Charge at Hydrophobic Uncharged Hydrophilic
Formula Mr Formula Mr pH 6.0–7.0 (nonpolar) (polar) (polar)
C3H7NO 2 89.1 C3H5NO 71.1 Neutral
C6H14N4O2 174.2 C6H12N4O 156.2 Basic (+ve)
C4H8N2O3 132.1 C 4 H 6 N2 O 2 114.1 Neutral
C4H7NO 4 133.1 C4H5NO3 115.1 Acidic(-ve)
C3H7NO2S 121.2 C3H5NOS 103.2 Neutral
C5H9NO 4 147.1 C5H7NO3 129.1 Acidic (-ve)
C5H10N2O3 146.1 C 5 H 8 N2 O 2 128.1 Neutral
C6H9N3O2 155.2 C6H7N3O 137.2 Basic (+ve)
C6H14N2O2 146.2 C6H12N2O 128.2 Basic(+ve)
C5H11NO 2S 149.2 C5H9NOS 131.2 Neutral
C3H7NO 3 105.1 C3H5NO2 87.1 Neutral
C11H12N2O2 204.2 C11H10N2O 186.2 Neutral

Appendix 9
Principles and standard conditions for
different purification techniques
Affinity chromatography (AC)
AC separates proteins on the basis of a reversible interaction between a protein (or group of proteins)
and a specific ligand attached to a chromatographic matrix. The technique is well-suited for a capture
or intermediate step and can be used whenever a suitable ligand is available for the protein(s) of interest.
AC offers high selectivity, hence high resolution, and usually high capacity [for the protein(s) of interest].
Affinity chromatography is frequently used as the first step (capture step) of a two-step purification
protocol, followed by a second chromatographic step (polishing step) to remove remaining impurities.
The target protein(s) is specifically and reversibly bound by a complementary binding substance
(ligand). The sample is applied under conditions that favor specific binding to the ligand. Unbound
material is washed away, and the bound target protein is recovered by changing conditions to those
favoring desorption. Desorption is performed specifically, using a competitive ligand, or nonspecifically,
by changing the pH, ionic strength, or polarity. Samples are concentrated during binding, and protein
is collected in purified and concentrated form. The key stages in an affinity chromatographic separation
are shown in Figure 98. Affinity chromatography is also used to remove specific contaminants; for
example, Benzamidine Sepharose 4 Fast Flow can remove serine proteases.
adsorption of wash elute
equilibration sample and away bound regeneration of media
elution of unbound protein(s)
unbound material material
Absorbance
begin sample change to

application elution buffer
>1
2 cv x cv 2–5 cv cv 2–3 cv
Column volumes [cv]
Fig 98. Typical affinity purification.
Further information
Protein Purification Handbook (Code No. 18-1132-29)
Affinity Chromatography Handbook: Principles and Methods (Code No. 18-1022-29)
Chapters 3 and 5 in this handbook for the purification of histidine- and GST-tagged proteins, respectively.
Ion exchange chromatography (IEX)

IEX separates proteins with differences in surface charge to give a very high resolution separation with
high sample loading capacity. The separation is based on the reversible interaction between a charged
protein and an oppositely charged chromatographic medium. Proteins bind as they are loaded onto a
column. Conditions are then altered so that bound substances are eluted differentially. Elution is usually
performed by increasing salt concentration or changing pH. Changes are made stepwise or with a
continuous gradient. Most commonly, samples are eluted with salt (NaCl), using a gradient elution (Fig 99).
Target proteins are concentrated during binding and collected in a purified, concentrated form.

sample gradient
equilibration wash reequilibration
application elution
high salt wash
1M
– 4 cv tightly bound
molecules
elute in high
unbound molecules elute
salt wash
before gradient begins
[NaCl]
10–20 cv
5 cv
5 cv
0
Column volumes [cv]
Fig 99. Typical IEX gradient elution.
The net surface charge of proteins varies according to the surrounding pH. Typically, when above its
isoelectric point (pI) a protein will bind to an anion exchanger; when below its pI a protein will bind to a
cation exchanger. However, it should be noted that binding depends on charge and that surface charges
may thus be sufficient for binding even on the other side of the pI. Typically IEX is used to bind the
target molecule, but it can also be used to bind impurities if required. IEX can be repeated at different
pH values to separate several proteins that have distinctly different charge properties, as shown in
Figure 100.
Selectivity
pH of mobile phase
Abs Abs Abs Abs
V V V V
+
Cation
Surface net charge
0 pH
Anion
Abs Abs Abs Abs
V V V V
Fig 100. Effect of pH on protein elution patterns.
Method development (in priority order)

1. Select optimal ion exchanger using small columns as in the HiTrap IEX Selection Kit to save time and sample.
2. Scout for optimal pH to maximize capacity and resolution. Begin 0.5 to 1 pH unit away from the
isoelectric point of the target protein if known.
3. Select the steepest gradient to give acceptable resolution at the selected pH.
4. Select the highest flow rate that maintains resolution and minimizes separation time. Check recommended
flow rates for the specific medium.

To reduce separation times and buffer consumption, transfer to a step elution after method
optimization as shown in Figure 101. It is often possible to increase sample loading when using
step elution.
high salt wash
elution 4 cv
of target
unbound elution of molecule
molecules unwanted 2–4 cv
elute material
sample
[NaCl]
injection tightly bound

volume molecules
2–4 cv elute
equilibration reequilibration
5 cv
5 cv
Column volumes [cv]
Fig 101. Step elution.
Further information
Ion Exchange Chromatography and Chromatofocusing Handbook: Principles and Methods
(Code No. 11-0004-21)
Hydrophobic interaction chromatography (HIC)

HIC separates proteins with differences in hydrophobicity. The technique is well-suited for the capture
or intermediate steps in a purification protocol. Separation is based on the reversible interaction between
a protein and the hydrophobic surface of a chromatographic medium. This interaction is enhanced by
high ionic strength buffer, which makes HIC an excellent “next step” after precipitation with ammonium
sulfate or elution in high salt during IEX. Samples in high ionic strength solution (e.g., 1.5 M ammonium
sulfate) bind as they are loaded onto a column. Conditions are then altered so that the bound substances
are eluted differentially.
Elution is usually performed by decreases in salt concentration (Fig 102). Changes are made stepwise
or with a continuous decreasing salt gradient. Most commonly, samples are eluted with a decreasing
gradient of ammonium sulfate. Target proteins are concentrated during binding and collected in a
purified and concentrated form. Other elution procedures include reducing eluent polarity (ethylene
glycol gradient up to 50%), adding chaotropic species (urea, guanidine hydrochloride) or detergents,
changing pH or temperature.
sample gradient reequilibration
equilibration salt free wash
application elution
1M
[ammonium sulfate]
tightly bound molecules

unbound molecules elute elute in salt free conditions
before gradient begins
10–15 cv
4 cv 5 cv
0
Column volumes [cv]
Fig 102. Typical HIC gradient elution.

Method development (in priority order)
1. The hydrophobic behavior of a protein is difficult to predict, and binding conditions must be studied
carefully. Use HiTrap HIC Selection Kit or RESOURCE HIC Test Kit to select the medium that gives optimal
binding and elution over the required range of salt concentration. For proteins with unknown hydrophobic
properties begin with 0–100%B (0%B, e.g., 1M ammonium sulfate). Knowledge about the solubility of the
protein in the binding buffer is important because high concentrations of, for example, ammonium
sulfate may precipitate proteins.
2. Select the gradient that gives acceptable resolution.
3. Select the highest flow rate that maintains resolution and minimizes separation time. Check recommended
flow rates for the specific medium.
4. If samples adsorb strongly to a medium then conditions that cause conformational changes, such as pH,
temperature, chaotropic ions, or organic solvents can be altered. Conformational changes caused by these
agents are specific to each protein. Use screening procedures to investigate the effects of these agents.
Alternatively, change to a less hydrophobic medium.
To reduce separation times and buffer consumption, transfer to a step elution after method
optimization, as shown in Figure 103. It is often possible to increase sample loading when using
step elution.
equilibration salt free wash reequilibration
elution of
unwanted elution
unbound 5 cv
material of target
molecules
molecule
[ammonium sulfate]
elute 2–4 cv
tightly bound
sample molecules
injection 2–4 cv elute
volume
5 cv
Column volumes [cv]
Fig 103. Step elution.
Further information
Hydrophobic Interaction Chromatography and Reversed Phase Handbook: Principles and Methods
(Code No. 11-0012-69)

Gel filtration (GF) chromatography
GF separates proteins with differences in molecular size. The technique is well-suited for the final
polishing steps in purification when sample volumes have been reduced (sample volume significantly
influences speed and resolution in gel filtration). Samples are eluted isocratically (single buffer, no
gradient, Fig 104). Buffer conditions are varied to suit the sample type or the requirements for further
purification, analysis, or storage, because buffer composition usually does not have major effects on
resolution. Proteins are collected in purified form in the chosen buffer.
high
molecular
weight
low
sample molecular
injection weight
volume
UV absorbance
intermediate
molecular weight
equilibration
1 cv
Column volumes (cv)
Fig 104. Typical GF elution.
Further information
Gel Filtration Handbook: Principles and Methods (Code No. 18-1022-18)
Reversed phase chromatography (RPC)

RPC separates proteins and peptides with differing hydrophobicity based on their reversible interaction
with the hydrophobic surface of a chromatographic medium. Samples bind as they are loaded onto a
column. Conditions are then altered so that the bound substances are eluted differentially. Due to the
nature of the reversed phase matrices, the binding is usually very strong. Binding may be modulated
by the use of organic solvents and other additives (ion pairing agents). Elution is usually performed by
increases in organic solvent concentration, most commonly acetonitrile. Samples, which are concen-
trated during the binding and separation process, are collected in a purified, concentrated form. The
key stages in a separation are shown in Figure 105.
column sample gradient clean after

reequilibration
equilibration application elution gradient
100% 2–4 cv
wash out unbound molecules

[CH 3 CN/0.1% TFA]
before elution begins
10–15 cv
5 cv
2 cv
0
Column volumes [cv]
Fig 105. Typical RPC gradient elution.

RPC is often used in the final polishing of oligonucleotides and peptides and is well-suited for analytical
separations, such as peptide mapping.
RPC is not recommended for protein purification if recovery of activity and return to a correct tertiary
structure are required, because many proteins are denatured in the presence of organic solvents.
Method development
1. Select medium from screening results.
2. Select optimal gradient to give acceptable resolution. For unknown samples begin with 0–100% elution
buffer.
3. Select highest flow rate that maintains resolution and minimizes separation time.
4. For large-scale purification transfer to a step elution.
5. Samples that adsorb strongly to a medium are more easily eluted by changing to a less hydrophobic medium.
Further information
Hydrophobic Interaction and Reversed Phase Chromatography Handbook: Principles and Methods
(Code No. 11-0012-69)

Product index
(Histidine)-tagged proteins Glutathione Sepharose 4B 105, 108, 109, 112, 113, 114–
119, 120, 125, 126, 138, 156,
Purification 163–165, 166,167, 211, 212,
Ni Sepharose High 214
Performance 23, 24, 25, 26, 28–29, 31–33,
GSTrap 4B 109, 113, 126–128, 131, 132,
39, 43, 45, 46, 97–98, 201, 202,
138, 139, 154–155, 167, 170,
203, 206
212–213
HisTrap HP 29, 46–47, 48, 49, 52–54, 92,
GST SpinTrap Purification
170, 187, 188–190, 203
Module 19, 107, 109, 113, 125, 154–155
His MultiTrap HP 12, 14, 15, 19, 27, 28–29, 39–41,
GST MultiTrap 4B 14, 15, 19, 107, 109, 113, 120–
202
123, 154–155, 212
His SpinTrap 19, 28–29, 43–45, 93, 99, 203
Ni Sepharose 6 Fast Flow 23, 24, 25, 26, 28–30, 34–38, Detection
39, 46, 55, 61, 66, 69, 70, 201, GST Detection Module 110, 113, 144–145, 151
202, 203, 204, 205, 206 GST 96-Well Detection Module 110, 142–144
HisTrap FF 28–30, 46–47, 50, 52–54, 103, Anti-GST Antibody 17, 110, 141, 142–143, 146,
170, 188–190, 203 148, 151
HisTrap FF crude 15, 27, 28–30, 55–57, 58, 59, Anti-GST HRP Conjugate 110, 148–149
60, 93, 104, 170, 188, 204
HisTrap FF crude Kit 28–30, 61–64, 65, 204 Tag cleavage
HisPrep FF 16/10 29–30, 50–51, 70–71, 170, 205 Enzymes
His MultiTrap FF 12, 14, 15, 19, 27, 28–30, 39–41, Thrombin 106, 110, 111, 140, 141, 153,
42, 202 154, 156–157, 160, 161, 162–165,
His GraviTrap 14, 15, 19, 27, 28–30, 66–68, 166, 167, 225
69, 93, 204 Factor Xa 106, 110, 111, 140, 141, 153,
His GraviTrap Kit 28–30, 66–68 154, 156–157, 162–165, 166,
167, 225
IMAC Sepharose High
Performance 25, 26, 72–73, 74–76, 80, 102, PreScission Protease 11, 91, 106, 110, 140, 141, 153,
207, 208, 210 154, 156–157, 158, 159, 162–164,
166, 167, 225
HiTrap IMAC HP 72–73, 80–82, 83, 102, 170,
208–209
Removal of thrombin
IMAC Sepharose 6 Fast Flow 25, 26, 72–73, 77–79, 80, 84,
87, 100–101, 170, 207, 208, and Factor Xa
209, 210 HiTrap Benzamidine FF
HiTrap IMAC FF 72–73, 80–82, 85–87, 100–101, (high sub) 111, 153, 154–155, 157, 160,
208–209 161, 163, 165, 167, 170
HiPrep IMAC FF 16/10 73, 84–85, 85–87, 170, 209 Benzamidine Sepharose 4
Fast Flow (high sub) 111, 228
His Buffer Kit 25, 30, 43, 44, 66
Detection Companion products
Anti-His antibody 69, 88, 89–90, 94 Western blotting
detection products 69, 88, 89–90,
GST-tagged proteins 110, 146–147, 148–149, 151,
Protein expression 152, 152, 167
pGEX vectors 105, 106, 107, 110–111, 138,
140, 144, 146, 148, 150, 158, Desalting and buffer
224–225 exchange
Purification HiTrap Desalting 26, 27, 33, 36, 63, 64, 76, 79,
101, 112, 113, 127, 132, 134,
Glutathione Sepharose
155, 162, 165, 166, 189, 191,
High Performance 105, 108, 109, 112, 114–119, 126,
192, 194–195, 196, 197, 215
127, 129, 138, 156, 163–165,
166, 167, 211, 212, 214 HiPrep 26/10 Desalting 26, 27, 33, 36, 63, 64, 76, 79,
86, 92, 101,104, 112, 127, 132,
GSTrap HP 109, 113, 126–128, 129, 132,
134, 162, 166, 189, 192, 196,
138–139, 154–155, 159, 167,
197, 198–199, 215
170, 212–213
PD-10 column 26, 27, 33, 34, 36, 37–38, 63,
Glutathione Sepharose 4
64, 76, 79, 101, 112, 127, 132,
Fast Flow 105, 108, 109, 112, 114–119,
134, 165, 166, 191, 192, 193,
120, 126, 134, 135, 138, 156,
215, 221
163–165, 166,167, 211, 212,
213, 214
GSTrap FF 109, 113, 126–128, 130, 132–133, HiLoad gel filtration
135–137, 138,139, 153, 154–155, products 60, 92, 103, 131, 159, 181, 182,
156–157, 158, 160, 161, 162–163, 183
167, 170, 212–213
GSTPrep FF 16/10 109, 113, 134–135, 135–137, Empty columns 31, 34, 74, 77, 98, 116, 117,
156, 170, 213 118, 181, 219, 220, 221, 223
GST MultiTrap FF 14, 15, 19, 107, 109, 113,
120–123, 124, 154–155, 212

Related literature
Code No.
Handbooks
GST Gene Fusion System 18-1157-58
Affinity Chromatography: Principles and Methods 18-1022-29
Gel Filtration: Principles and Methods 18-1022-18
Hydrophobic Interaction and Reversed Phase Chromatography: Principles and Methods 11-0012-69
Ion Exchange Chromatography and Chromatofocusing: Principles and Methods 11-0004-21
Protein Purification 18-1132-29
Challenging Protein Purification 28-9095-31
2-D Electrophoresis 80-6429-60
Selection guides/brochures
Ni Sepharose and IMAC Sepharose, selection guide 28-4070-92
Affinity Columns and Media, selection guide 18-1121-86
Convenient Protein Purification, HiTrap column guide 18-1129-81
Gel Filtration Columns and Media, selection guide 18-1124-19
Ion Exchange Columns and Media, selection guide 18-1127-31
Prepacked chromatography columns with ÄKTAdesign systems, selection guide 18-1173-49
Protein purification—applications that meet your needs, application brochure 11-0027-81
CD
Column Packing CD—The Movie 18-1165-33
The Protein Purifier—Software-based learning aid for purification strategies 18-1155-49
Data files and application notes

Ni Sepharose 6 Fast Flow, HisTrap FF, and HisPrep FF 16/10 columns 11-0008-86
Ni Sepharose High Performance and HisTrap HP columns 18-1174-40
HisTrap FF crude columns and HisTrap FF crude Kit 11-0012-37
His GraviTrap 11-0036-90
His MultiTrap FF and His MultiTrap HP 11-0036-63
His SpinTrap 28-4046-59
IMAC Sepharose 6 Fast Flow, HiTrap IMAC FF, and HiPrep IMAC FF 16/10 columns 28-4041-06
IMAC Sepharose High Performance and HiTrap IMAC HP columns 28-4041-05
Glutathione Sepharose High Performance and GSTrap HP columns 18-1174-32
Glutathione Sepharose 4 Fast Flow, GSTPrep FF 16/10, and GSTrap FF 18-1136-89
GSTrap 4B columns 28-4048-14
GST MultiTrap FF, GST MultiTrap 4B 28-4081-57
Addition of imidazole during binding improves purity of histidine-tagged proteins 28-4067-41

Ordering information
Product Quantity Code No.
Histidine-tagged proteins
Purification
Ni Sepharose High Performance 25 ml 17-5268-01
100 ml* 17-5268-02
HisTrap HP 5 × 1 ml 17-5247-01
100 × 1 ml† 17-5247-05
1 × 5 ml 17-5248-01
5 × 5 ml 17-5248-02
100 × 5 ml† 17-5248-05
His MultiTrap HP 4 × 96-well filter plates 28-4009-89
His SpinTrap 50 × 100 µl 28-4013-53
Ni Sepharose 6 Fast Flow 5 ml 17-5318-06
25 ml 17-5318-01
100 ml 17-5318-02
500 ml* 17-5318-03
HisTrap FF 5 × 1 ml 17-5319-01
100 × 1 ml† 17-5319-02
5 × 5 ml 17-5255-01
100 × 5 ml† 17-5255-02
HisTrap FF crude 5 × 1 ml 11-0004-58
100 × 1 ml† 11-0004-59
5 × 5 ml 17-5286-01
100 × 5 ml† 17-5286-02
HisTrap FF crude Kit 3 × 1 ml, buffers 28-4014-77
HisPrep FF 16/10 1 × 20 ml 17-5256-01
His MultiTrap FF 4 × 96-well filter plates 28-4009-90
His GraviTrap 10 × 1 ml 11-0033-99
His GraviTrap Kit 20 × 1 ml, buffers 28-4015-51
IMAC Sepharose High Performance 25 ml 17-0920-06
100 ml* 17-0920-07
HiTrap IMAC HP 5 x 1 ml 17-0920-03
5 x 5 ml 17-0920-05
IMAC Sepharose 6 Fast Flow 25 ml 17-0921-07
100 ml* 17-0921-08
HiTrap IMAC FF 5 x 1 ml 17-0921-02
5 x 5 ml 17-0921-04
HiPrep IMAC FF 16/10 1 x 20 ml 17-0921-06
His Buffer Kit 11-0034-00
HiTrap Chelating HP 5 × 1 ml 17-0408-01
1 × 5 ml 17-0409-01
5 x 5 ml 17-0409-03
100 x 5 ml† 17-0409-05
Chelating Sepharose Fast Flow 50 ml 17-0575-01
500 ml* 17-0575-02
Detection
Anti-His antibody 170 µl 27-4710-01

GST-tagged proteins
Protein expression
pGEX- 4T-1 25 µg 27-4580-01
pGEX- 4T-2 25 µg 27-4581-01
pGEX- 4T-3 25 µg 27-4583-01
pGEX- 5X-1 25 µg 27-4584-01
pGEX- 5X-2 25 µg 27-4585-01
pGEX- 5X-3 25 µg 27-4586-01
pGEX- 6P-1 25 µg 27-4597-01
pGEX- 6P-2 25 µg 27-4598-01
pGEX- 6P-3 25 µg 27-4599-01
All vectors include E. coli B21
Purification
Glutathione Sepharose High Performance 25 ml 17-0579-01
100 ml* 17-0579-02
GSTrap HP 5 × 1 ml 17-5281-01
100 × 1 ml† 17-5281-05
1 × 5 ml 17-5282-01
5 × 5 ml 17-5282-02
100 × 5 ml† 17-5282-05
Glutathione Sepharose 4 Fast Flow 25 ml 17-5132-01
100 ml 17-5132-02
500 ml* 17-5132-03
GSTrap FF 2 × 1 ml 17-5130-02
5 × 1 ml 17-5130-01
100 × 1 ml† 17-5130-05
1 × 5 ml 17-5131-01
5 × 5 ml 17-5131-02
100 x 5 ml† 17-5131-05
GSTPrep FF 16/10 1 × 20 ml 17-5234-01
GST MultiTrap FF 4 × 96-well filter plates 28-4055-01
Glutathione Sepharose 4B 10 ml 17-0756-01
100 ml (function tested) 27-4574-01
300 ml* 17-0756-04
GSTrap 4B 5 × 1 ml 28-4017-45
100 × 1 ml† 28-4017-46
1 × 5 ml 28-4017-47
5 × 5 ml 28-4017-48
100 × 5 ml† 28-4017-49
GST SpinTrap Purification Module 50 × 50 µl 27-4570-03
GST MultiTrap 4B 4 x 96-well filter plates 28-4055-00
Detection
GST Detection Module 50 detections 27-4590-01
GST 96-Well Detection Module 5 plates 27-4592-01
Anti-GST Antibody 0.5 ml, 50 detections 27-4577-01
Anti-GST HRP Conjugate 75 µl RPN1236

Tag cleavage
Enzymes
Thrombin 500 units 27-0846-01
Factor Xa 400 units 27-0849-01
PreScission Protease 500 units 27-0843-01
Removal of thrombin and Factor Xa

HiTrap Benzamidine FF (high sub) 2 × 1 ml 17-5143-02
5 × 1 ml 17-5143-01
1 × 5 ml 17-5144-01
Benzamidine Sepharose 4 Fast Flow (high sub) 25 ml* 17-5123-10
Companion products
E. coli B21 1 vial 27-1542-01
Isopropyl β-D-thiogalactoside (IPTG) 1g 27-3054-03
5g 27-3054-04
10 g 27-3054-05
Western blotting
Hybond-P 10 sheets RPN2020F
Hybond-ECL 10 sheets RPN2020D
ECL Western Blotting
Anti-GST HRP Conjugate 75 µl RPN1236
ECL GST Western Blotting Detection Kit 1 kit RPN1237
2
Detection Reagents for 1000 cm RPN2109
ECL Plus Western Blotting
Detection System for 1000 cm2 RPN2132

PD-10 Desalting Columns 30 17-0851-01
HiTrap Desalting 5 × 5 ml 17-1408-01
100 x 5 ml† 17-1408-05
HiPrep 26/10 Desalting 1 × 53 ml 17-5087-01
4 x 53 ml 17-5087-02
Gel filtration
HiLoad 16/60 Superdex 30 pg 1 x 120 ml 17-1139-01
1 x 320 ml 17-1140-01
1 x 320 ml 17-1070-01
1 x 320 ml 17-1071-01
HiPrep 16/60 Sephacryl S-100 HR 1 x 120 ml 17-1165-01
1 x 320 ml 17-1194-01
1 x 320 ml 17-1195-01
1 x 320 ml 17-1196-01
* Larger quantities are available; please contact GE Healthcare.
† Special pack size delivered on specific customer order

Empty columns
Complete information on the range of Tricorn columns is available
at www.gehealthcare.com/protein-purification-labresearch
Tricorn 5/100 column 1 18-1163-10
Tricorn 5/150 column 1 18-1163-11
Tricorn 5/200 column 1 18-1163-12
Tricorn 10/100 column 1 18-1163-15
Tricorn 10/150 column 1 18-1163-16
Tricorn 10/200 column 1 18-1163-17
Tricorn columns are delivered with a column tube, adaptor unit,
end cap, a filter kit containing adaptor and bottom filters and O-rings,
two stop plugs, two fingertight fittings, adaptor lock and filter holder,
and two M6 connectors for connection to FPLC™ System, if required.
XK 16/20 column 1 18-8773-01
XK 26/20 column 1 18-1000-72
XK 50/20 column 1 18-1000-71
XK columns are delivered with one AK adaptor, TEFZEL tubing
(0.8 mm i.d. for XK 16 and XK 26 columns, 1.2 mm i.d. for XK 50 columns,
with M6 connectors, thermostatic jacket, support snap-on net rings,
dismantling tool (XK 16 and XK 26 only), and instructions.
HR 16/5 column 1 18-1000-98
HR 16/10 column 1 19-7403-01
HR 16/50 column 1 18-1460-01
HR columns are delivered with a column tube, adaptor unit, end cap,
a filter kit containing adaptor and bottom filters and O-rings and
M6 male fittings for connection to FPLC System.
Empty disposable PD-10 Desalting columns 50 17-0435-01
Accessories and spare parts

For a complete listing refer to GE Healthcare BioDirectory or
www.gehealthcare.com/protein-purification
LabMate PD-10 Buffer Reservoir 10 18-3216-03
Packing Connector XK 16 1 18-1153-44
Packing Connector XK 26 1 18-1153-45
Tricorn packing equipment 10/100
Tricorn packing equipment 10/100 includes Tricorn packing connector
10-10, Tricorn 10/100 glass tube, bottom unit and stop plug. 1 18-1153-25
Tricorn packing connector 10-10‡ 1 18-1153-23
Connects extra glass column to a Tricorn 10 column to
act as a packing reservoir for efficient packing.
1/16” male/Luer female‡ 2 18-1112-51
Tubing connector flangeless/M6 female‡ 2 18-1003-68
‡
Tubing connector flangeless/M6 male 2 18-1017-98
Union 1/16” female/M6 male‡ 6 18-1112-57
Union M6 female /1/16” male 5 18-3858-01
Union Luerlock female/M6 female 2 18-1027-12
HiTrap/HiPrep, 1/16” male connector for ÄKTAdesign 8 28-4010-81
Stop plug female, 1/16”§ 5 11-0004-64
Fingertight stop plug, 1/16”¶ 5 11-0003-55
‡ One connector included in each HiTrap package
§
Two, five, or seven female stop plugs included in HiTrap packages, depending on products
¶
One fingertight stop plug is connected to the top of each HiTrap column

Handbooks
from GE Healthcare
Protein Purification GST Gene Fusion System

Handbook Handbook
18-1132-29 18-1157-58
Gel Filtration Hydrophobic Interaction and

Principles and Methods Reversed Phase Chromatography
18-1022-18 Principles and Methods
11-0012-69
Affinity Chromatography
Principles and Methods 2-D Electrophoresis
18-1022-29 using immobilized pH gradients
Handbook
18-1037-46 Microcarrier Cell Culture
Percoll 18-1140-62
Methodology and Applications
18-1115-69 Challenging Protein Purification
Handbook
Ion Exchange Chromatography 28-9095-31
& Chromatofocusing
Principles and Methods Recombinant Protein
11-0004-21 Purification Handbook
18-1142-75
GE Healthcare
Recombinant Protein Purification Handbook – Principles and Methods

ÄKTA, ÄKTAdesign, ÄKTAcrossflow, ÄKTAexplorer, ÄKTAFPLC, ÄKTApilot ,
www.gehealthcare.com/protein-purification ÄKTAprime, ÄKTAprocess, ÄKTApurifier, ÄKTAxpress, AxiChrom, ECL Advance,
ECL Plus, ExcelGel, FPLC, GraviTrap, GSTPrep, GSTrap, HiLoad, HiPrep, HisPrep,
www.gehealthcare.com/his HisTrap, HiTrap, Hybond, Labmate, MabSelect, MabSelect SuRe, MabSelect Xtra,
Mono Q, Multiphor, MultiTrap, NAP, Percoll, PhastGel, PhastSystem, PreScission,
PrimeView, Rainbow, RESOURCE, Sephadex, SpinTrap, Sephacryl, Sepharose,
GE Healthcare Bio-Sciences AB SOURCE, Superdex, Superloop, Tricorn, UNICORN and Drop design are trademarks
of GE Healthcare companies. GE, imagination at work, and GE monogram are
Björkgatan 30 trademarks of General Electric Company.
Tricorn column and components are protected by US design patents USD500856,
751 84 Uppsala USD506261, USD500555, USD495060 and their equivalents in other countries.
US patent numbers 5,284,933 and 5,310,663, and equivalent patents and patent
Sweden applications in other countries (assignee: Hoffman La Roche, Inc) relate to the
purification and preparation of fusion proteins and affinity peptides comprising at
least two adjacent histidine residues (commonly known as the histidine-tag
technology). Any customer that wishes to use Chelating Sepharose Fast Flow,
Ni Sepharose 6 Fast Flow, or IMAC Sepharose 6 Fast Flow for non-research/
commercial applications under these patents is requested to contact Hoffman-
La Roche AG, Corporate licensing, attention Dr. Andreas Maurer, CH-4070 Basel,
Switzerland, telephone +41 61 687 2548, fax +41 61 687 2113, for the purpose of
obtaining a license.
PGEX Vectors are to be used for scientific investigation and research and for no
other purpose whatsoever, and a license for commercial use of the licensed
products and the processes claimed in the patents must be negotiated directly
with Chemicon International Inc by the purchaser prior to such use.
Any use of UNICORN software is subject to GE Healthcare Standard Software
End-User License Agreement for Life Sciences Software Products. A copy of this
Standard Software End-User License Agreement is available on request .
All third party trademarks are the property of their respective owners.
© 2000–2006 General Electric Company – All rights reserved.
First published Dec 2000.
All goods and services are sold subject to the terms and conditions of sale of the
company within GE Healthcare that supplies them. A copy of these terms and
conditions is available on request . Contact your local GE Healthcare
representative for the most current information.
GE Healthcare Europe GmbH
Munzinger Strasse 5
Recombinant Protein
D-79111 Freiburg, Germany
GE Healthcare UK Limited
Amersham Place
Little Chalfont
Buckinghamshire, HP7 9NA, UK
GE Healthcare Bio-Sciences Corp.
800 Centennial Avenue
P.O. Box 1327
Piscataway, NJ 08855-1327, USA
GE Healthcare Bio-Sciences KK
Sanken Bldg.3-25-1
Hyakunincho Shinjuku-ku
Tokyo 169-0073, Japan
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18-1142-75 AC 01/2007

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GE Healthcare

Recombinant Protein Purification Handbook – Principles and Methods

Protein Purification GST Gene Fusion System

Gel Filtration Hydrophobic Interaction and

Gel filtration Hydrophobic interaction Ion exchange Affinity Reversed phase

Fig 1. Separation principles in chromatographic purification.

highlights chemicals, buffers and equipment.

outline of experimental protocol.

Pure tagged Pure denatured

cell protein Pure tagged

Processing Bacteria Yeast Insect Mammalian

methods for yeast than for E. coli.

Histidine tag GST tag

Insoluble in Soluble in Periplasmic Culture

Cell lysis Cell wall disruption

Cell debris removal Cell removal Cell removal

Harvest inclusion Recover Recover clarified Recover clarified

Fig 4. Overview of sample preparation.

Extraction process Typical conditions Comment

Extraction should be performed quickly, at sub-ambient temperatures, in the presence of a

Nominal pore size of filter Particle size of chromatographic medium

Remove salts from proteins with molecular weight Mr > 5000.

Detection and quantitation

Assessing protein expression

Analytical tool Characteristic being assessed

Tagged recombinant proteins for simple purification

Manual purification techniques

Fig 5. ÄKTAprime plus. Fig 6. ÄKTAexplorer.

Fig 7. Four modules of ÄKTAxpress system.

Standard ÄKTAdesign configurations

ÄKTAprime plus ÄKTApurifier ÄKTAexplorer ÄKTAxpress

ÄKTApilot ÄKTAcrossflow ÄKTAprocess

Pure tagged Pure denatured

cell protein Pure tagged

Working with nickel-containing products may produce an allergic reaction.

We recommend binding at neutral to slightly alkaline pH (pH 7 to 8) in the presence of 0.5 to

Alternative elution solutions

General procedure for sample preparation

If the recombinant histidine-tagged protein is expressed as inclusion bodies, they must be

Selection Guide — Precharged Ni Sepharose products

Will you use

Batch Ni Sepharose 6 Fast Flow

Syringe 96-well His MultiTrap FF

Spin His SpinTrap

HisTrap FF crude Kit Contains Ni Sepharose High Performance.

Fig 10. Selection guide for the precharged Ni Sepharose products.

Product Format Approx. Description High- Mini- Batch/ Syr- ÄKTAdesign

Unclarified HisTrap FF crude

Product Format Approx. Description High- Mini- Batch/ Syr- ÄKTAdesign

Product Format Approx. Description High- Mini- Batch/ Syr- ÄKTAdesign

Ni Sepharose is compatible with reducing agents. However, we recommend removal of any

Ni Sepharose is compatible with reducing agents. However, we recommend removal of any

Preparing the empty Disposable PD-10 Column

Ni Sepharose is compatible with reducing agents. However, we recommend removal of any

Centrifugation procedure for high-throughput screening

Preparing the filter plate

A) B) FC12 DDM Cymal 6 TX-100 FC12 DDM Cymal 6 TX-100

UDM Cymal 5 OG LDAO UDM Cymal 5 OG LDAO

Equilibrate column Apply sample Elute