gel electrophoresis

1. RAMABAI BARLINGAY
SCHOOL OF
BIOTECHNOLOGY
Name- MUGDHA RANGNATH
Class- M.Sc. Biotechnology- Semester 1
Seminar Topic:-
GEL ELECTROPHORESIS

2. INTRODUCTION
Electrophoresis is the migration of charged particles of
molecules in medium under the influence of an applied
field. This method was developed by Arne W.K. Tiselius in 1937. The usual purpose of carrying out
electrophoretic experiments are –
 To determine the number, amount and mobility of components in given sample or to separate them.
 To determine molecular weight of protein in DNA, RNA sequence.
Types of electrophoresis are -
• Free electrophoresis.
• Zone electrophoresis.
Gel electrophoresis is one of the general technique of zone electrophoresis. It is the process that separates
fragments of DNA based on their sizes.

3. GEL ELECTROPHORESIS
Charge on molecule is the major determinant for its electrophoretic mobility and ultimate
separation from rest of the molecules.

4.  Starch gel - Potato starch is hydrolyzed in acidified acetone where temperature and timing of
hydrolyses is extremely important. The suspension is then neutralized with sodium acetate and
washed with large amount of distilled water and dried acetone. The hydrolyzed starch when heated
and cooled in an appropriate buffer sets as a gel. In starch gel the amylopectin chains are intertwine.
Specially used for analysis of isoenzymes patterns
 Polyacrylamide gel – The most components to be used for preparation of this gel are acrylamide
monomer, NN’- methylenebisacrylamide, ammonium persulphate and tetramethylenediamine
(TEMED) .
The types of gels are :-

5. It has low absorption capacity, suitable in qualitative analysis. The pore size of the gel is determined
by the amount of acylamide used per unit volume of the reaction medium and the degree of cross
linkage, which is dependent upon the amount bisacrylamide used. It is poured vertically
 Agar gel - It consists of two galactose based poymers i.e. agarose and agaropectin. The agaropectin is
suphated and hence it is charged and may give rise to electrophoresis which may interfere with electrophoretic
separation . The sulphate is removed by purification. A solution of agar is mixed with polyethylene glycol. The
prepcipitate is collected and washed and dried with acetone. The agar is added to aqueous buffer. High
concentration of agar gives rise to smaller pore size. It is used to separate macromolecules like proteins and
nucleic acid.
 Agarose gel - Agarose is produced from agar. These gels are hydrophilic. They are almost completely inert to
the solute being separated. Agarose has greater porosity and used to separate moleclues upto molecular weight
several million daltons. High salt concentration salts also do not affect the gel but higher temperature may alter
their structure. Is is poured horizontally.
 Agarose- Acrylamide gel - Acrylamide gels with pores large enough remains almost liqiud because the quantity
of acrylamide is very less. It has no molecular seiving action necessary for sharp resolution. Acrylamide gel
provides flexibilty for sharp resolution.

6. Components & Requirements of an
Electrophoresis System
 An electrophoresis chamber and high
voltage power supply of generally 0.1-6
kV.
 Combs  it is used to put wells in the cast
gel for sample loading gel.
 Gel casting tray  the open ends of trays
are closed with tape while the gel is being
cast, the removed prior to electrophoresis.
 Agarose gel  a porous material that
molecules migrate through.
 Buffer  a fluid mixture of water and ions
(buffer should also be unreactive).

7. Instrumentation
 A typical setup consists of a gel slab sandwiched between two glass plates, with the ends
enclosed in upper and lower reservoirs of buffer.
 Samples to be run are loaded in wells at the top of gel.
 An electric voltage is applied between the upper and lower reservoirs causing samples to
migrate down. The voltages used for electrophoresis are sufficient to cause electrocoution .
Electromotive force moves molecules through the matrix of different speeds based on size
or charge.
 Samples used - Samples may be materials containing protiens or nucleic acids. The may
be biologically derived for example prokaryotic or eukaryotic cells, tissues, viruses,
environmental samples, or purified protiens.

8. Preparation of gel and buffer:-
The top layer is stacking gel bottom layer is separating gel.
 Stacking gel:- Tris HCL buffer; pH- 6.8.
To concentrate all the protiens in the sample into a thin narrow band at the top of the resolving gel. It also
contains wells into which the samples are loaded. Cl ions are small, negatively charged and moves
quickly through the gel.
 Separating gel (resolving gel):- Tris HCL buffer; pH- 8.8.
It performs the actual separation of the protien. Macromlocules separates according to their size. The
resolving gel is samll pore PAG.
 Running buffer:- Tris/ Glycine. SDS is used for denaturation of sample.
 Sample buffer (glycerol or sucrose):- It is used to obtain higher viscosity of the gel. Glycerol is
used to make the sample denser than sample buffer so the sample will remain in the bottom of the well
rather than float out.

9. Procedure---
 Prepare gel.
 Place the gel tray into casting apparatus and add gel. Allow it to solidify.
 Tape the open edges of gel tray and place comb into gel to create wells.
 Add enough running buffer to cover the surface of the gel.
 Slowly and carefully load samples into gel.
 An appropriate marker should be loaded along with experimental samples.
 Turn the power on.
 Run the gel until the dye has
migrated to the appropriate distance.
 Remove gel from cassette, drop gel into
staining dish.
 Excess dye is washed and gel is destained.

10. Working
 When the power is switched on we see the
amount of bubbles generated in the reservoir
containing the cathode (upper reservoir) and less amount at anode (lower reservoir).
 The anode, to which negatively charged macromolecules would migrate under an electric field is
placed in lower buffer reservoir. Here SDS gives each protein a negative charge proportionate to its
mass. Since they have equal charge due to SDS, they will migrate in the gel on the basis of their
molecular weight.
 Buffers in gels provided ions to carry a current and maintain pH . The buffer is allowed to enter drop
by drop to avoid short circuit. If electrophoresis is going to take long time it is necessary to maintain
buffer in upper reservoir.
 Charged molecules loaded in gel move into the gel through the electrical attraction by opposite
charged pole.
 Higher the voltage the more quickly the gel runs and rate of
migration increases.

11.  Direction of protein migration is from cathode to anode.
 When the dye reaches at anodic end of
electrophoresis medium, electrophoresis is
stopped. Before removing the gel, power must be
turned off and cables removed.
 Tracking Dye (Staining)
• After completion of electrophoresis, gel is
introduced in the staining solution.
• Bromophenol blue is used for monitoring the
electrophoretic run.
• The dye migrates faster than all
macromolecules.
• The extent of migration of the dye gives an
index of electrophoretic process.

12.  Destaining
• The dye actually penetrates the entire gel, however it sticks permanently to the proteins.
• Destaining solution is hence used to get the display pattern of blue protein band against
the clear background.
• The destaining solution can be prepared from methanol, glacial acetic acid and distilled
water in appropriate quantity.
• At first wash solution shrinks the gel, squeezing out much of the liquid component, and
gel swells and clears in second wash. Destain the gels until the bands are properly seen.
• Properly stained/destained gels can be dried and photographed for later analysis.
Fluorescence under UV lights makes bands visible.

13. Agarose gel electrophoresis
 Agarose gel electrophoresis is a method of gel electrophoresis used to separate a mixed population of
macromolecules such as DNA or proteins in matrix of agarose.
 This is achieved by moving negatively charged nucleic acid molecules with an electric filed.
 Agarose gels has large pore size and good gel strength and it is suitable for electrophoresis of DNA and
large protein molecules.
 Most agarose gels used are between 0.7-2% dissolved in suitable electrophoresis buffer.
 Agarose gel electrophoresis can resolve DNA fragments that range roughly from 500 to 30,000 base
pairs.

14. PAGE
 Polyacrylamide Gel Electrophoresis describes
a technique widely used in biochemistry,
forensics, genetics, molecular biology and
biotechnology to separate biological
macromolecules, usually proteins or nucleic
acids, according to their electrophorectic
mobility.
 Mobility is a function of the length,
conformation and charge of the molecule.
 Molecules may run in their native state,
preserving the molecules’ higher-order
structure. This method is called Native-PAGE.
 Alternatively, a chemical denaturant may be
added to remove this structure and turn the
molecule into an unstructured molecule whose
mobility depends only on its length and mass-
to-charge ratio. This procedure is called SDS.

15. SDS-PAGE
 SDS-PAGE is the most widely used method for analyzing protein mixture qualitatively. Is specially
useful as separation is based completely on relative molecular weight of protein.
 The samples are treated with Sodium Dodecyl Sulfate an anionic detergent. It is composed of a
hydrophilic group with a net negative charge and long hydrophobic chain with neutral charge. Hence it
imparts equal negative charge to all samples and denatures protein by breaking the disulfide bonds
in proportion its mass.
 SDS binds to hydrophobic region of the protein and can separate the components of proteins.
 The number of SDS molecules that binds is proportional to the size of the protein, thereby in the
electric field, protein molecules move towards anode (+ electrode) and separated only according to
their molecular weight.
 Without SDS different proteins with similar molecular weights would migrate differently due
differences in folding patterns and it would cause some proteins to better fit through the gel matrix than
others. SDS linearizes the proteins so that they may be strictly separated by molecular weight.
 A concurrent treatment with a disulfide reducing agent as mercaptoethanol which further denatures
the proteins and breaking up their structures.
 Protein separation by SDS-PAGE can be used to estimate relative molecular mass, to determine the
relative abundance of major proteins in a sample, and to determine the distribution of proteins
among proteins.

18. • Principles and techniques of biochemistry and molecular biology by Keith Wilson
and John Walker.
• Biophysical techniques by Upadhahyay & Nath.
• Fundamentals of biochemistry by J.L. Jain.
• Fundamentals and Techniques of Biophysics and Molecular Biology by Pranav
Kumar.
ThankYou!