Vertical Gel Electrophoresis

Raghavendra Institute of Pharmaceutical Education and Research - Autonomous
K.R.Palli Cross, Chiyyedu, Anantapuramu, A. P- 515721
Vertical Gel Electrophoresis
A Seminar as a part of curricular requirement
for M. Pharmacy, I Year - I semester
Presented by
SHAIK FIRDOUS BANU
(20L81S0105)
PHARMACOLOGY
Under the guidance of
Dr. P. Ramalingam M.Pharm, Ph.D.
Professor & Director of R& D cell

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K.R.Palli Cross, Chiyyedu, Anantapuramu, A. P- 515721 2
Contents
 Introduction
 Vertical gel electrophoresis
 Principle
 2D Polyacrylamide Gel Electrophoresis
 Applications
 Advantages and disadvantages
 References

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Introduction:
• Gel electrophoresis is a method for separation and analysis of
macromolecules like DNA, RNA and protein or their
fragments, based on their size and charge.
• Based on gel casting technique, classified in to :
1) Horizontal Gel Electrophoresis.
2) Vertical Gel Electrophoresis.

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 Cross-linked polyacrylamide
gels are formed from the
polymerization of acrylamide
monomer in the presence of
smaller amount of
methylenebisacrylamide.
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Vertical gel electrophoresis

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Principle:
• Any charged molecule or ion migrate when placed in an
electric field, the rate of migration depends upon its
net charge, size, shape and the applied electric current.
• Can be represented by following equation:
V=E* q
f
Where,
V=velocity of migration of the molecule
E=electric field
q=net electric charge on the molecule
f=frictional coefficient
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Types of PAGE
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PAGE can be classified according the separation conditions
into:
• Native-PAGE:
• Native gels are run in non-denaturing conditions, so that the
analyte’s natural structure is maintained.
• Separation is based upon charge, size and shape of
macromolecules.
• Useful for separation and purification of mixture of proteins.

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SDS PAGE :
• When Sodium dodecyl sulfate detergent SDS
added to PAGE the combined procedure is termed as
SDS PAGE.
• It has strong protein denaturing effect.
• SDS coats proteins molecules giving all proteins a constant charge
mass ratio.
• Proteins migrate along the gel in order of increasing size or
molecular weights.
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Gradient PAGE
• Unlike fixed concentration gels, gradient gels are
formulated with a range of polyacrylamide concentrations.
• Where the gradient begins with a lower concentration and ends
with a higher concentration.
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Importance of Gradient PAGE
• A much greater range of protein can be separated
than on a fixed –percentage gel.
• Proteins with similar molecular weight can be resolved.
• The higher the polyacrylamide gels, the smaller the
pore Size in the matrix.
• Higher concentration gels can separate small sized proteins.
• While low concentration gels, with larger pore size are better
at resolving higher molecular weight proteins.
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Gradient gel preparation
• It uses two chambers, where one contains the acrylamide
solution at the lowest gradient concentration, and the other
contain the higher acrylamide Concentration.
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• Stacking gel (4%):
It concentrate all the proteins in one band, so that they
will start migrating in running gel all at the same time.
• Running gel (12%) :
Running gel allows to separate the Proteins based on their
molecular weight.
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Running the gel
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• Take out the gels from the casting frame and clamp them
in the gel apparatus.
• When the plates are secured, place them in cassette and
then lock.
• Place them in the gel running tank.
• Fill the inner chamber of the tank with buffer.
• Remove the comb carefully.

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• Microgram quantity of the sample is placed over the
top of the gel column.
• Cathode and anode are kept above and below the
Column to impose an electric field through the column.
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• There is no external solvent space, all the migratory
particles have to pass through the gel pores.
• Different sample components get separated in to discrete
migratory bands along the gel column on the basis of
electrophoretic mobility and gel filtration effect.
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Visualization
• Ethidium bromide or coomassie blue dye may be used
for this process.
• If the analyte molecule
fluoresce under the UV light,
a photograph can be taken
of the gel under UV lighting
conditions.
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2D Polyacrylamide Gel Electrophoresis
Principle :
• Proteins were resolved on a gel using isoelectric
focusing(IEF) in first dimension.
• In second dimension in the Presence of sodium dodecyl
sulfate.
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First dimension : Isoelectric Focusing
Isoelectric point :
It is defined as the PH of a solution at which
the net charge of the protein become zero.
• A protein with positive net charge migrate towards cathode
becoming less positively charged until it reaches it Pi.
• While a protein with a negative net Charge migrate towards
anode becoming less negatively charged until it reaches it Pi.
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• A protein mixture is loaded at the basic end of
the PH Gradient gel.
• Large proteins will move more
Slowly through the gel, but with
sufficient time will catch up with
Small proteins of equal charge.
• These IPG Strips placed in
SDS Gel.
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Applications of polyacrylamide gel electrophoresis:
• Used for estimation of molecular weight of protiens and nucleic acids.
• Determination of subunit structure of protien.
• Purification of isolated proteins.
• Monitoring changes of proteins contents in body fluids.
• Identifying disulfide bonds between protein
• Quantifying proteins
• Blotting applications
• Comparison of polypeptide composition of different samples.

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• Advantages of Polyacrylamide Gel Electrophoresis:
• Stable chemically cross-linked gel.
• Greater resolving power.
• Good for separation of low molecular weight fragments.
• Pore size of the acrylamide gels can be altered in an easy and
controllable fashion by changing the concentration of the
two monomers.
• Disadvantages:
• Generally more difficult to prepare and handle, involving
longer time for preparation than agarose gel.
• Need new gel for each experiment .

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Reference:
• John M. Walker. Gradient SDS Polyacrylamide
Gel Electrophoresis of Proteins. Springer. 1994; 32(7): 35-38.
• Bryan John Smith. SDS Polyacrylamide Gel Electrophoresis of
Proteins. Springer. 1994; 32: 23-34.
• Jaap H. Waterborg, Harry R. Matthews. The Electrophoretic
Elution of Proteins from Polyacrylamide Gels. Springer. 1994;
32: 169-175.
• Paula Meleady. Two –Dimensional Gel Electrophoresis. Springer.
2017; 1664: 3-14.
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THANK YOU
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Vertical Gel Electrophoresis

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