Electrophoresis: by Vasudha Saswati Jyotsna Priyanka

ELECTROPHORESIS
By
Vasudha
Saswati
Jyotsna
Priyanka
ELECTROPHORESIS
DEFINITION: It describes migration of charged

particles or molecules under the influence of electric
field.
PURPOSE FOR CARRYING OUT

ELECTROPHORESIS
1. To determine the number, amount and mobility of
components in a given sample or to separate
them.
2. To obtain information about the electrical double
layers surrounding the particles.
3. Determination of molecular weight of proteins and
DNA sequencing.
PRINCIPLE
Any charged ion or molecule migrates when
placed in an electric field. The rate of
migration depend upon its net charge, size,
shape and the applied electric current.
Can be represented by following eq.
E*q
v = ---------------
f
 v = velocity of migration of the molecule.
 E = electric field in volts per cm
 q = net electric charge on the molecule
 f = frictional coefficient
The movement of charged particle in an electric field is

expressed in terms of electrophoretic mobility ,denoted by µ.
where,
µ = v/E OR
µ = q/f
For molecules with similar conformation f varies with size but not
with shape. Thus electrophoretic mobility (µ) of a molecule is
directly proportional to charge density(charge\mass ratio).
FACTORS AFFECTING
ELECTROPHORETIC MOBILITY
1. Charge – higher the charge greater the
electrophoretic mobility.
2. Size – bigger the molecule greater are the

frictional and electrostatic forces exerted on it by
the medium. Consequently, larger particles have
smaller electrophoretic mobility compared to
smaller particles.
3. Shape – rounded contours elicit lesser frictional

and electrostatic retardation compared to sharp
contours. Therefore globular protein move faster
than fibrous protein.
TYPES OF
ELECTROPHORESIS:
ELECTROPHORESIS
FREE ZONE
ELECTROPHORESIS ELECTROPHORESIS
MICRO MOVING PAPER CELLULOSE ACTTATE GEL

ELECTROPHORESIS BOUNDARY ELECTROPHORESIS ELECTROPHORESIS ELECTROPHORESIS
PAPER ELECTROPHORESIS
It is the form of electrophoresis that is carried out on
filter paper. This technique is useful for separation
of small charged molecules such as amino acids
and small proteins.
FILTER PAPER : It is the stabilizing medium. We

can use whatman filter paper, cellulose acetate
filter paper or chromatography paper.
APPARATUS : Power pack, electrophoretic cell that
contains electrodes, buffer reservoirs, support for
paper, transparent insulating cover.
SAMPLE APPLICATION : The sample may be applied as a
spot(about 0.5 cm in diameter)or as a uniform streak.
ELECTROPHORETIC RUN:The current is switched on after
the sample has been applied to the paper and the paper has
been equilibrated with the buffer.The types of buffer used
depends upon the type of separation.Once removed, the paper
is dried in vaccum oven.
DETECTION AND QUANTITATIVE ASSAY:To identify
unknown components in the resolved mixture the
electrophoretogram may be compared with another
electrophoretogram on which standard components have been
electrophoresced under identical conditions.Physical
properties like fluorescence,ultraviolet absorption or
radioactivity are exploited for detection.
GEL
ELECTROPHORESIS
It is a technique used for the separation of Deoxyribonucleic
acid, Ribonucleic acid or protein molecules according to their
size and electrical charge using an electric current applied to a
gel matrix.
What is a gel?
Gel is a cross linked polymer whose composition and porosity is
chosen based on the specific weight and porosity of the target
molecules.
Types of Gel:
 Agarose gel.
 Polyacrylamide gel.
AGAROSE GEL
 A highly purified uncharged polysaccharide derived from
agar.
 Used to separate macromolecules such as nucleic
acids, large proteins and protein complexes.
 It is prepared by dissolving 0.5% agarose in boiling
water and allowing it to cool to 40°C.
 It is fragile because of the formation of weak hydrogen
bonds and hydrophobic bonds.
POLYACRYLAMIDE GEL
 Commonly used components: Acrylamide monomers,

Ammonium persulphate, Tetramethylenediamine
(TEMED), N,N’-methylenebisacrylamide.
 These free radicals activate acrylamide monomers
inducing them to react with other acrylamide monomers
forming long chains.
 Used to separate most proteins and small
oligonucleotides because of the presence of small pores.
ELECTROPHORESIS OF PROTEINS
The most commonly used technique for the separation of
proteins is Sodium dodecyl sulphate-polyacrylamide gel
electrophoresis (SDS PAGE).
PROCEDURE
 Protein sample is first boiled for 5 mins in a buffer solution
containing SDS and β-mercaptoethanol.
 Protein gets denatured and opens up into rod-shaped
structure.
 Sample buffer contains bromophenol blue which is used as
a tracking dye, and sucrose or glycerol.
 Before the sample is loaded into the main separating gel a
stacking gel is poured on top of the separating gel.
PROCEDURE Continued…
 Current is switched on.
 The negatively charged protein-SDS complexes now
continue to move towards the anode.
 As they pass through the separating gel, the proteins
separate, owing to the molecular sieving properties of the
gel.
 When the dye reaches the bottom of the gel, the current is
turned off.
 Gel is removed from between the glass plates and shaken
in an appropriate stain solution.
 Blue colored bands are observed under UV rays.
CONTINUOUS AND DISCONTINUOUS
BUFFER SYSTEMS
 A continuous buffer system has only a single separating gel

and uses the same buffer in the tanks and the gel.
 In a discontinuous system a non-restrictive large pore gel,

called a stacking gel, is layered on top of a separating gel.
E.g. SDS PAGE Electrophoresis.
 The resolution obtainable in a discontinuous system is

much greater than that obtainable in a continuous one.
However the continuous system is easier to set up.
ISOELECTRIC FOCUSING
 Electrophoretic method that separates
proteins according to the iso-electric points
 Is ideal for seperation of amphoteric
substances
 Seperation is achieved by applying a
potential difference across a gel that contain
a pH gradient
 Isoelectric focusing requires solid support
such as agarose gel and polyacrylamide gel
 Isoelectric focusing gels contain synthetic
buffers called ampholytes that smooth the
pH gradients.
 Ampholytes are complex mixtures of
synthetic polyamino-polycarboxylic acids
 Commercially available ampholytes are-
BIO-LYTE
PHARMALYTE
 It gives good separation with a high
resolution compared to any other method
 Resolution depends on
I. The pH gradient,
II. The thickness of the gel
III. Time of electrophoresis,
IV. The applied voltage,
V. Diffusion of the protein into the gel.
PREPARATION OF IEF GEL
A TYPICAL ISOELECTRIC
FOCUSING GEL
TWO-DIMENSIONAL
ELECTROPHORESIS
 This technique combines the technique IEF
(first dimension), which separates proteins in
a mixture according to charge (PI), with the
size separation technique of SDS-PAGE
second dimension).
 The combination of these two technique to

give two-dimension(2-D)PAGE provides a
highly sophisticated analytical method for
analysing protein mixtures.
 To maximise separation, most workers use

large format 2-D gels(20cm x 20cm).
 Although the minigel system can be used
to provide useful separation in some cases.
 For large-format gels,the first

dimension(isoelectric focusing) is carried
out in an acrylamide gel that has been cast
on a plastic strip(18cm x3mm wide).
 The gel contains ampholytes (for forming

pH gradient) together with 8M urea and a
non-ionic detergent, both of which denature
and maintain the solubility of the proteins
being analysed.
 The denatured proteins therefore separate in this gel
according to their isoelectric points.
 The IEF strip then incubated in a sample buffer
containing SDS (thus binding SDS to the denatured
proteins) and then placed between the glass plates of a
previously prepared 10% SDS-PAGE gel.
 Electrophoresis is commenced and the SDS-bound
proteins run into the gel and separate according to size.
 The IEF gels are provided as dried strips and need
rehydrating overnight.
 The first dimension IEF run then takes 6-8h, the
equilibration step with SDS sample buffer takes about
15 min, and then the SDS-PAGE step takes about 5h.
 Using this method one can routinely resolve
between 1000 and 3000 proteins from a cell or
tissue extract and in some cases workers have
reported the separation of between 5000 and
10000 proteins.
 The result of this is a gel with proteins spread

out on its surface. These proteins can then be
detected by a variety of means, but the most
commonly used stains are silver and coomasie
staining.
2D-gel (coomassie stained)
Thank you

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ELECTROPHORESIS

DEFINITION: It describes migration of charged

PURPOSE FOR CARRYING OUT

The movement of charged particle in an electric field is

2. Size – bigger the molecule greater are the

3. Shape – rounded contours elicit lesser frictional

MICRO MOVING PAPER CELLULOSE ACTTATE GEL

FILTER PAPER : It is the stabilizing medium. We

 Commonly used components: Acrylamide monomers,

 A continuous buffer system has only a single separating gel

 In a discontinuous system a non-restrictive large pore gel,

 The resolution obtainable in a discontinuous system is

 The combination of these two technique to

 To maximise separation, most workers use

 For large-format gels,the first

 The gel contains ampholytes (for forming

 The result of this is a gel with proteins spread

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