1. Submitted by,
ABEL JACOB THOMAS
MSc 2nd SEMESTER
CFT-k

2.  Electrophoresis is the migration of charged particles or molecules in a
medium under the influence of an applied electric field.
 It originated from Greek words, ‘Electro’ meaning electric flow of current
and ‘Phoresis” meaning movement. Thus it means transport by electricity.
 It can be used to separate :
Proteins in body fluids like serum, urine
Proteins in erythrocytes like hemoglobin
Nucleic acids like DNA, RNA
 Depending on the charge the molecule carry, they move either towards the
cathode or to the anode. An ampholyte become positively charged in acidic
medium and migrate to cathode and in alkaline condition they become
negatively charged and migrate towards anode.

3.  The rate of migration of an ion in electric field depends on factors like :
1. Net charge of molecule
2. Size and shape of particle
3. Strength of electrical field
4. Properties of supporting medium
5. Temperature of operation
 Principle of electrophoresis – There are two electrodes, which are made of
an inert material like platinum, are immersed in two separate buffer
chambers. These chambers are not fully separated from each other. By
using electrical power supply, electrical potential E is generated between
two electrodes. Due to potential, electrons move from anode to cathode.
Hence anode become positively charged and cathode become negative.
Electrons driven to cathode leave the electrode and take part in reduction
reaction with water generating H₂ gas and hydroxide ions. While
oxidation take place in anode. Electrons released from water enter the
electrode generating O₂ gas and free protons. Amount of electrons leaving
cathode equals amount of electrons entering cathode. Thus negatively charged anions
move to positive anode and positively charged cations move to negative cathode.

4.  Factors affecting electrophoresis
1. The sample – Charge/mass ratio of sample dictates its electrophoretic
mobility.
2. The electric field – An increase in the potential gradient increases the
rate of migration.
3. The medium – The inert medium can exert adsorption and molecular
sieving effects on the particle, influencing the rate of migration.
4. The buffer – the composition, ionic strength and pH can affect the
electrophoretic mobility of the sample in different ways.

5. 1. Free solution electrophoresis – No stabilizing media used.
They are mainly of ;
a) Moving boundary electrophoresis
b) Free flow electrophoresis
c) Micro electrophoresis
d) Capillary electrophoresis
e) Microchip electrophoresis
2. Zone electrophoresis – Migration of charged particles on supporting media.
Paper, cellulose acetate membrane, starch gel, polyacrylamide gel etc are
used. They are mainly of ;
a) Paper electrophoresis
b) Cellulose acetate electrophoresis
c) Gel electrophoresis

6.  Gel electrophoresis – This is a molecular sieving technique based on
molecular size of the substances. The gel acts as a molecular sieve.
 The supporting media used here must be electrically neutral. They can be
agar and agarose gel, starch etc.
 The porous gel acts as a sieve by retarding or in some cases completely
obstructing the movement of macromolecules while allowing smaller
molecules to migrate freely.
 Gel electrophoresis can be of three main types :
a) Starch gel electrophoresis
b) Agarose gel electrophoresis
c) Polyacrylamide gel electrophoresis [ PAGE ]
 Polyacrylamide gel electrophoresis – It is used for the separation and
analysis of proteins and relatively small nucleic acid molecules.
 The resolution of PAGE is so high in the size range of about 10 – 1000
nucleotide units, it is capable of separating DNA molecules that differ in
length by a single monomer unit.

7.  PAGE methods are of different types like :
a) SDS – PAGE
b) Isoelectric focusing
c) 2D PAGE
 These methods are applied mostly for the separation of proteins based on
distinct molecular properties. Different proteins have different shapes and
sizes, and also during electrophoresis, proteins are separated by complex
combination of their charge, shape, size. The separation of proteins have
high resolution.
 2D or native PAGE is an electrophoresis method to separate native
proteins. Here the migrating proteins are kept in their native state by
applying conditions. The buffers provide non-denaturing native state and
process is carried out in low temperature to dissipate heat.
 2D PAGE is also useful in checking uniformity of the isolated protein. Even
if the purified protein sample contain single type of protein, they might not
be uniform. They must have been unfolded or have undergone chemical
modification. 2D PAGE can also detect complex formation between
proteins. These complex can be detected as an extra band.

8.  It is an electrophoresis method to separate proteins.
 Developed by Ulrich K Laemmli
 Separates proteins with molecular masses between 5 and 250 KDa.
 Proteins migrate in their denatured state
 The migration velocity of protein is a function of their size shape, and the
number of electric charges they carry. The native PAGE cant be used to
estimate molecular mass of proteins. They also are unable to assess
whether a purified protein is composed of single subunit or multiple
subunits. Even a multi- subunit protein may migrate as a single sharp
band. SDS-PAGE was introduced to analyse such cases and to allow the
estimation of molecular mass of single subunit proteins or those of
individual subunits of multi subunit proteins.

9.  SDS ( Sodium Dodecyl Sulphate) is an anionic detergent. It is the
polyacrylamide based discontinuous gel used here.
 The SDS polyacrylamide gel is typically sandwiched between two glass
plates in a slab gel. Although tube gels in glass cylinders were used
historically, they were rapidly made obsolete with the invention of more
convenient slab gels.
 SDS acts as a surfactant, masking the protein’s intrinsic charge and
conferring them very similar charge to mass ratios. The intrinsic charge of
proteins are negligible in comparison to SDS loading, and the positive
charges are also greatly reduced in the basic pH range of a separate gel.
 Upon application of a constant electric field, the protein migrate towards
the anode, each with a different speed, depending on its mass. This simple
procedure allows precise protein separation by mass.
 SDS tends to form spherical micelles in aqueous solution and above a
certain concentration called the critical micellar concentration (CMC).
 Above CMC of 7-10 mill molar in solutions, SDS simultaneously forms
single monomer and as micelles.
 Below CMC it occurs only as monomers in aqueous solutions.
 At CMC, a micelle consist of a micelle consist of about 62 SDS molecules.

11.  Only SDS monomers bind to proteins via hydrophobic interactions, where
as SDS micelles are anionic on the outside and don't absorb any protein.
 SDS is amphipathic in nature, which allows to unfold both polar and non-
polar sections of protein structure.
 In SDS concentrations of above 0.1mM, the unfolding of proteins begins
and above 1mM, most proteins are denatured.
 Due to strong denaturing effect of SDS, and the subsequent dissociation of
protein complexes, quaternary structures can generally not be determined
with SDS. Exceptions are proteins that are stabilized by covalent cross
linking like disulphide linkages and the SDS resistant protein complexes,
which are stable even in the presence of SDS. To denature the SDS
resistant complexes, a high activation energy is needed, which is achieved
by heating.
 Thus, when proteins are treated with SDS at high temperatures, radical
conformational changes occur. This treatment breaks all native non-
covalent intermolecular and intramolecular interactions. The subunit
structure or multi subunit proteins disintegrates and the proteins unfold.

12.  SDS molecules bind to unfolded proteins in large excess, providing large
extra negative charges to the molecules. The amount of bound SDS
molecules is largely independent of the amino acid sequence of the
polypeptide chain and is roughly linear function of the polypeptide length.
 Therefore upon SDS treatment, the specific charge of different protein
becomes identical and also the shape of different molecules become similar.
 The negatively charged SDS molecules repeat each other, which lends a rod
like shape to the SDS treated proteins.
 Thus SDS treated proteins Just like denatured linear DNA molecules, will
be separated solely by their size.
 As size is a linear function of mass, SDS-PAGE ultimately separates
proteins based on their molecular mass.
 The relative mobility, i.e. the running distance of protein divided by
running distance of the tracking dye of SDS treated protein is inversely
proportional to logarithm of molecular mass of protein.
 By running several proteins of known molecular mass alongside the
proteins of interest, a log molecular mass – relative mobility calibration
curve can be drawn and based on this curve, molecular mass of unknown
protein can be calculated.

13. 1. SAMPLE PREPARATION
40µL of protein sample + 10µL of disruption buffer
Boil the mixture ( 99℃ for 3 min )
 Disruption buffer contains –
• 10% (w/v) SDS
• 1M Tris – HCl, pH 6.8
• Glycerol
• β mercaptoethanol
• Bromophenol blue

14. 2. POLYACRYLAMIDE GEL PREPARATION
Acrylamide gel is prepared – Cross linked polyacrylamide gels are formed
from the polymerization of acrylamide monomer in the presence of small
amount of N,N’–methylene–bisacrylamide. Polyacrylamide is chemically
inert, electrically neutral, hydrophilic, transparent for optical detection.
Preparation and setting up –
i. Clean the plates and combs
ii. Set up the plates on the racks
iii. Pour the separation gel
iv. Pour the stacking gel
v. Gel storage
Protein samples are loaded in the stacking layer. Purpose of stacking layer is
to get all protein samples lined up so they can enter the separation layer
at exactly the same time. Both stacking and separating gel are
polyacrylamide bt stacking has lower conc. of polyacrylamide. Stacking gel
pH is 6.8 and that of separating is 8.8.Stacking gel is on the top of
separating gel.

15. 3. Running the gel using running buffer
Running gel contains Tris – HCl – glycene – SDS
i. Boil the samples completely to denature proteins
ii. Assemble gel into apparatus
iii. Pour the running buffer solution in to the chamber
iv. Load 20µL of samples into the well
v. Run electrophoresis by connecting the current supplies.
4. Stain the gel using staining buffer - Used for the visualization of protein
bands. This visualizes the band under UV light. Coomassie blue or silver
stain is used.
5. De – staining the gel using de – staining buffer – Contains glacial acetic
acid, methanol

18.  Applications
• Determine purity of protein samples
• Determine molecular weight of protein
• Identifying disulfide bonds between protein
• Quantifying proteins
 Advantages
• Migration is directly proportional to molecular weight
• Highly sensitive test, separates even 2% difference in mass
• Require only small amount of sample
• Stable chemically cross – linked gel

19.  Disadvantages
• Poor band resolution due to high alkaline operating pH
• Acrylamide gel is potent neurotoxin chemical
• Gel preparation is difficult and require longer time
• High cost

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scientific publishers, New Delhi. 1999
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Delhi. 2002