Electrophoresis:

Electrophoresis is the migration of charged particles or molecules in a medium under the

influence of an applied electric field.
Principle: Many important biological molecules such as amino acids, peptides, proteins,
nucleotides and amino acids possess ionizable groups and exist electrically charge species
(either as cation or anions) at given pH. Under the influence of an electrical field these charge
particles, cations (+), migrate to the cathode or anions (-) move to the anode, depending on
the nature of their net charge. Separation of sample depends on charge, size and shape.
The rate of migration of an ion in electrical field depend on factors:

 Net charge of molecule: The grater the number of charges on a particle, grater the relative
distance migrated by charge particle
 Size and shape of particle: When net charge is equal, smaller particles move faster rate than
large particle
 pH of buffer: The direction of movement of charge particles depends on pH of the buffer.
 Strength of electrical field: By increasing the applied voltage, the migration distance of the
charge particles can be increased
 Properties of supporting medium: Migration rate of compounds depends upon type of
supporting medium. Inert medium is always preferred. The medium might cause adsorption,
molecular sieving, and electro-osmosis processes that affect the electrophoretic rate.
Adsorption causes tailing of the sample, leading to movement of sample in the form of comet
rather than a band. This reduces rate as well as resolution of the separation.
 Temperature of operation: Temperature plays a bit role in electrophoresis. With decreasing
the temperature your band become more dense or neatly packed in gel.
 Ionic strength of buffer : When ionic strength of a buffer is increased , decrease in the
migration of charge particle take place. High ionic strength of the buffer lead to increase
heat generation. This leads to increase in the rates of diffusion of ions and also denaturation
of sample materials like proteins and enzyme. Buffer ionic strength should be in the range of
0.025 to 1 in electrophoretic separation.

Basic component of electrophoresis

1. Electrophoresis chamber : All the process take place. There are two type of chamber
a) Vertical : it is used for separating proteins in polyacrylamide gel
b) Horizontal : It is used for electrophoresis of serum, Hb,
immunoelectrophoresis, isoelectric focusing and for DNA & RNA in
agarose
2. Electric Power Supply : It is a source of constant voltage or current that provides
energy to the electrode. This drives the movement of ions in the medium and results
in the movement and separation of molecules in the specimen. The desired
voltage/current can be set according to the need of test.
3. Buffer medium: Buffer chosen in that way that effective separation takes place. It
maintains the constant state of ionization of the molecules being separated. Also
determine the net charge of the molecules. Buffer carry the current during
 electrophoresis and maintain pH during electrophoresis. Most of the protein, the
electrophoresis is performed at pH 8.6.
4. Supporting Medium: It is selected to ensure accurate and effective separation. The
sample is placed on supportive medium. Supportive medium should contain buffer
for separation.
i. It may be paper in paper electrophoresis
ii. Gel in gel electrophoresis
Kind of supportive medium used :

 Agarose / Agar : Generally agarose is used 0.5 to 2 % for electrophoresis.

Percentage of agarose depends on size of molecules to be separated. Fot
the separation of larger molecules low % of agarose should be used
 Polyacrylamide: Acrylamide, N, N'-Methylene bisacrylamide, Ammonium
Persulfate and TEMED
 Cellulose acetate: The pores of cellulose acetate paper are homogenous
and standardized . This is very delicate in compare to paper. It gives
minimum absorption and clear separation of sample. Excellent
supporting medium for the separation of serum proteins, hemoglobulin.
Separation takes less time.
 Cellulose: Filter paper (Whatman 1 mm or 3 mm) is very cheap and easy
to use. Separation takes long time. On paper undesirable
electroendosmosis effects are seen. Now it is largely replaced by other
supporting media.

The principle and method of polyacrylamide gel electrophoresis (SDS-PAGE)

When proteins are separated by electrophoresis through a gel matrix, smaller proteins
migrate faster due to less resistance from the gel matrix. Other influences on the rate of
migration through the gel matrix include the structure and charge of the proteins.
In SDS-PAGE, the use of sodium dodecyl sulfate (SDS, also known as sodium lauryl sulfate) and
polyacrylamide gel largely eliminates the influence of the structure and charge, and proteins
are separated solely based on polypeptide chain length.
SDS is a detergent with a strong protein-denaturing effect and binds to the protein backbone
at a constant molar ratio. In the presence of SDS and a reducing agent that cleaves disulfide
bonds critical for proper folding, proteins unfold into linear chains with negative charge
proportional to the polypeptide chain length.
.
Polymerized acrylamide (polyacrylamide) forms a mesh-like matrix suitable for the
separation of proteins of typical size. The strength of the gel allows easy handling.
Polyacrylamide gel electrophoresis of SDS-treated proteins allows researchers to separate
proteins based on their length in an easy, inexpensive, and relatively accurate manner.
Material Required:
1) 40% Acrylamide (37.5:1): Acrylamide, N, N'-Methylene bisacrylamide
2) 30% Ammonium Persulfate: Ammonium Persulfate
3) TEMED
4) Buffer--1.5 M Tris*C1, pH 8.8
5) Buffer--1.0 M Tris•Cl, pH 6.8
6) 4x SDS-PAGE Sample Buffer: 125 mM Tris•HC1, pH 6.8 , 4% Glycerol, 4% SDS ,1% B-
Mercaptoethanol ,0.5 mg/ml Bromophenol Blue
7) Running Buffer pH 8.3: Tris base, Glycine, SDS
8) Coomassie Stain Solution: Ethanol 30%, Glacial Acetic Acid 10%, Coomasie Brilliant
Blue—R-250 : 0.2%
9) Destain Solution: Ethanol 30%, Glacial Acetic Acid 10%,

Casting the Gel

 Stacking Gel 4% : Tris chloride buffer pH 6.8, Acrylamide, SDS, Ammonium

persulphate & TEMED
 Running Gel 8%: Tris chloride buffer pH 8.8, Acrylamide, SDS, Ammonium
persulphate & TEMED

i. Assemble glass plates and spacers in gel casting apparatus

ii. Mix the components for the resolving gel.
iii. Pour the resolving gel mixture into the gel plates to a level 2 cm below the top of the
shorter plate
iv) Pace a layer of DDI H2O over the top of the resolving gel to prevent meniscus
formation in the resolving gel.
v) Allow resolving gel to stand 30 min at room temperature.
vi) Drain the D H2O from top of the resolving gel. Rinse with D H2O, drain, and wick any
remaining DH2O away with a Kimwipe.
vii) Mix components for stacking gel.
viii) Pour stacking gel solution into gel plates (on top of running gel), so that gel plates
are filled. Insert comb to the top of the spacers.
ix) Allow gel to stand for at least 1 hr at room temperature, or overnight at 4°C
(wrapped in saran wrap).

Running the Gel

• Remove comb and assemble cast gel into Mini-Protean II apparatus.
• Add freshly prepared lx running buffer (300 ml) to both chambers of the apparatus.
 Load the prepared samples into the wells of the gel.
• Run the gel at 100 V until the dye front migrates into the running gel (15 min), and
increase to 200 V until the dye front reaches the bottom of the gel ( 45 min).

Staining & Distaining the Gel

 Remove the run gel from the aparatus and remove the spacers and glass plates.
Place the gel into a small tray. Note: Never us a metal spatula to separate the glass
plates.
 Add , 20 ml staining solution and stain for > 30 min with gentle shaking. Pour off and
save the stain.
 Add , 5 ml destain solution and destain for —1 min with gentle shaking.
 Pour off and discard the destain solution. Add — 30 ml of destain solution. Destain
with gentle shaking until the gel is visibly destained (> 2 hr).
 Pour off and discard the destain solution.
 Rinse with DDI H2O. Add —30 ml DDI H2O and rinse for 5 min with gentle shaking.
 Dry the gel on the gel dryer at 60°C for 1 hr with a sheet of Whatman filter paper
below the gel and a piece of Seran wrap over the gel.
 Electrophoresis of DNA
Agarose gel electrophoresis is commonly used to separate DNA fragments following
restriction endonuclease digestion or PCR amplification. DNA and RNA are negatively charged
and during electrophoresis, the side of the gel having wells is placed near the cathode. The
negatively charge DNA moves toward anode under electric current/voltage.
Tris-acetate-EDTA or tris-borate-EDTA (TBE) buffers are used for DNA/RNA electrophoresis.
Bromophenol blue or xylene cyanol are used as loading dye and mixed with the nucleic acid
sample so that, the electrophoretic run can be tracked till these dyes move near the other
end. Fragments are detected by staining the gel with the intercalating dye, ethidium bromide,
followed by visualization/photography under ultraviolet light.
Material required:
i) Horizontal electrophoresis apparatus
ii) UV transilluminator
iii) Agarose
iv) Tris Acetate EDTA buffer (TAE, 50X) or TBE buffer 10X)
v) Loading Dye (6X)
vi) Ethidium bromide (EtBr)
Preparation of the Gel:
1. Weigh out the appropriate mass of agarose into an Erlenmeyer flask. Agarose gels
are prepared using a w/v percentage solution. The concentration of agarose in a gel
will depend on the sizes of the DNA fragments to be separated, with most gels
ranging between 0.5%-2%. The volume of the buffer should not be greater than 1/3
of the capacity of the flask.
2. Add running buffer ( TAE to the agarose-containing flask. Swirl to mix. The most
common gel running buffers are TAE (40 mM Tris-acetate, 1 mM EDTA) and TBE (45
mM Tris-borate, 1 mM EDTA).
3. Melt the agarose/buffer mixture. This is most commonly done by heating in a
microwave, but can also be done over a Bunsen flame. At 30 s intervals, remove the
flask and swirl the contents to mix well. Repeat until the agarose has completely
dissolved. Cool down to 45 to 50 degree celcious.
4. Add ethidium bromide (EtBr) to a concentration of 0.5 μg/ml. Alternatively, the gel
may also be stained after electrophoresis in running buffer containing 0.5 μg/ml EtBr
for 15-30 min,
Note: EtBr is a suspected carcinogen and must be properly disposed of per institution
regulations. Gloves should always be worn when handling gels containing EtBr.
Alternative dyes for the staining of DNA are available; however EtBr remains the most
popular one due to its sensitivity and cost.
Casting of Gel:
 i. Place the gel tray into the casting apparatus. Alternatively, one may also tape the open
edges of a gel tray to create a mold. Place an appropriate comb into the gel mold to
create the wells.
ii. Pour the molten agarose into the gel mold. Allow the agarose to set at room
temperature. Remove the comb and place the gel in the electrophoresis apparatus .
Setting up of Gel Apparatus and Separation of DNA Fragments:

 Add loading dye to the DNA samples to be separated (Fig. 2). Gel loading dye is
typically made at 6X concentration (0.25% bromphenol blue, 0.25% xylene cyanol,
30% glycerol). Loading dye helps to track how far your DNA sample has traveled, and
also allows the sample to sink into the gel.
 Program the power supply to desired voltage (1-5V/cm between electrodes).
 Add enough running buffer to cover the surface of the gel. It is important to use the
same running buffer as the one used to prepare the gel.
 Attach the leads of the electrophoresis apparatus to the power supply. Turn on the
power supply and verify that both gel box and power supply are working.
 Remove the lid. Slowly and carefully load the DNA sample(s) into the gel. An
appropriate DNA size marker should always be loaded along with experimental
samples.
 Replace the lid to the electrophoresis apparatus. The cathode (black leads) should be
closer the wells than the anode (red leads). Double check that the electrodes are
plugged into the correct slots in the power supply.
 Turn on the power. Run the gel until the dye has migrated to an appropriate
distance.
 Observing Separated DNA fragments
 When electrophoresis has completed, turn off the power supply and remove the lid
of the gel box.
 Remove gel from the electrophoresis apparatus. Drain off excess buffer from the
surface of the gel. Place the gel tray on paper towels to absorb any extra running
buffer.
 Remove the gel from the gel tray and place it on UV transilluminator & expose the
gel to uv light (wave length 312nm) . This is most commonly done using a gel
documentation system. DNA bands should show up as orange fluorescent bands.