Gel electrophoresis is a method to separate biomolecules like proteins, nucleic acids, and lipids based on their charge and size. During gel electrophoresis, an electric current is applied across a gel, causing negatively charged molecules to migrate toward the positive electrode and positively charged molecules to migrate toward the negative electrode. Smaller molecules migrate faster through the gel than larger molecules. Factors like the charge, size, and shape of molecules, as well as the electric current, gel composition, and buffer used, determine how far each type of molecule will migrate through the gel. Gel electrophoresis has applications in separating DNA, RNA, proteins, and other biomolecules.

1. Chethan. N
1st M Pharm
Dept. of Pharmacology,
J.S.S.C.P,
MYSORE.

2.  Positive or negative electrical charges are
frequently associated with biomolecules. When
placed in an electric field, charged biomolecules
move towards the electrode of opposite charge due
to the phenomenon of electrostatic attraction
Introduction:

3. Electrophoresis
 Electrophoresis is the separation of charged
molecules in an applied electric field.
 The relative mobility of individual molecule
depends on several factors. The most important of
which are:
 Net charge
 Charge/mass ratio,
 Molecular shape and
 The temperature, porosity and viscosity of the matrix through
which the molecule migrates.

4. Gel Electrophoresis
 Gel electrophoresis is a method for separation and
analysis of macromolecules like DNA, RNA and
proteins or their fragments, based on their size and
charge.
 Gel electrophoresis uses a gel as an anti-convective
medium and/or sieving medium during
electrophoresis.
 Gels suppress the thermal convection caused by
application of the electric field, gels can also simply
serve to maintain the finished separation, so that a
post electrophoresis stain can be applied.

5. Principle
 By placing the substance to be separated in wells of the gel
and applying an electric current, allows the molecule to move
through the matrix at different rates towards the anode if
negatively charged or toward the cathode if positively
charged.
 As they move through the gel, the larger molecules will be
held up as they try to pass through the pores of the gel, while
the smaller molecules will be impeded less and move faster.
 This results in a separation by size, with the larger molecules
nearer the well and the smaller molecules farther away.

6. Principle of separation
 According to charge: When charged molecules are
placed in an electric field, they migrate toward either
the positive (anode) or negative (cathode) pole
according to their charge.
 According to size: The smaller molecules move more
swiftly than the larger sized ones, as the can travel
through the pores more easily than the later.

7. Instrument and reagents
1. Electrophoresis apparatus
2. Buffer
3. Power supply
4. Supporting media
5. Detection and Quantification

8. 1.Electrophoresis apparatus:
•The casting tray is
made up of glass or
plastic.
•The comb contains
varying number of
teeth in order to help
in formation of well.

9. Electrophoresis apparatus set up:
•Electrophoresis
chamber with buffer
solution
•Casting tray
•Electrodes

10. 2. Buffer:
 Buffers in gel electrophoresis are used to provide ions
that carry a current and to maintain the pH at a
relatively constant value.
 The most common being, for nucleic acids
Tris/Acetate/EDTA (TAE), Tris/Borate/EDTA(TBE).

11. 3. Power supply:
• The electrodes are
connected to their
respective terminals of
the electrophoresis
chamber and to the
power supplier with
controls for rate of
current flow.
•The best resolution of
fragments larger than
about 2 kb is attained by
applying no more than 5
volts per cm to the gel

12. 4. Supporting media: (Gel)
1. Starch
2. Agar/agarose
3. Cellulose acetate
4. Polyacrylamide gel
 The kind of supporting matrix used depends on type
of molecules to be separated and the desired basis for
separation: charge, molecular weight or both

13.  Agarose and polyacrylamide gels are cross-linked,
spongelike structure
 It is important that the support media is electrically
neutral. Presence of charge group may cause:
 -Migration retardation
 -The flow of water toward one or the other electrode
so called ‘Electroendosmosis (EEO)’, which decrease
resolution of the separation
 Agarose Gels have fairly large pore sizes and are used
for separating larger DNA molecules (Restriction
Fragment Length Polymorphism Analysis)
 Polyacrylamide Gels are used to obtain high resolution
separations for smaller DNA molecules (STR analysis
and DNA sequence analysis)

14. 5. Detection and quantification:
 Stains
 Protein staining
 Ethidium bromide staining
 Blotting
 Southern blotting (for DNA)
 Northern blotting (for RNA)
 Western blotting (for protein)

15. Ethidium bromide Protein staining

16. Process of gel electrophoresis

17. Gel electrophoresis
Agarose gel
- electrode + electrodeDNA fragments
buffer~~~~~~~~~~~~~~~~~~~~~~~~ ~~~~~~~~~~~~~~~~~~~~~~~~
~~~~~~~~~~~~~~~~~~~~~~~~ ~~~~~~~~~~~~~~~~~~~~~~~~
- electrode + electrode
current
buffer~~~~~~~~~~~~~~~~~~~~~~~~
~~~~~~~~~~~~~~~~~~~~~~~~ ~~~~~~~~~~~~~~~~~~~~~~~~
~~~~~~~~~~~~~~~~~~~~~~~~

18. visualization
 The molecules in the gel are stained to make them visible.
DNA may be visualized using ethidium bromide which, when
intercalated into DNA, fluoresce under ultraviolet light, while
protein may be visualised using silver stain or Coomassie
Brilliant Blue dye.
 SYBR Green I is more expensive, but 25 times more sensitive
and possible safer than ethidium bromide.
 SYBR Safe is a variant of SYBR Green, and show low level of
mutagenicity and toxicity.
 Other less frequently used markers are Cresol red and Orange
G.

19. Factors affecting separation in gel
electrophoresis
 The sample:
 The charge/mass ratio of the sample dictates its
electrophoretic mobility.
 Charge: Higher the charge, greater the electrophoretic
mobility.
 Size: Size is inversely proportional to electrophoretic
mobility.
 Shape: Globular substances move faster than the
fibrous ones.

20.  The electric field: An increase in the potential
gradient increases the rate of migration.
 The medium: The inert medium can exert
adsorption or molecular sieving effects on the
particle influencng its rate of migration.
 Adsorption: retention of the component on the
surface of supporting medium.
 Molecular sieving: media such as agar,
polyacrylamide, sephadex have cross linked
structures giving rise to pores within the gel
beads.

21.  The buffer: the buffer can affect the electrophoretic
mobility by:
 Ionic strength: increase in ionic strength of buffer
means a larger share of current is carried by buffer and
smaller proportion by sample, while decrease in ionic
strength is vice-versa.
 pH: pH determines the degree of ionization of organic
compounds. Where ionization is inversely proportional
to pH.

22. Applications
 Separation of Deoxyribonucleic acid
 Separation of ribonucleic acid
 Separation of protein molecules
 It may be used as preparative technique prior to use of other
methods such as mass spectroscopy, cloning, DNA Sequences,
Southern Blotting for further characterization.
 Separation of amino acid
 Separation of lipoproteins
 Separation of enzyme in blood
 Separation of antibiotic drug

23. References:
 Instrumental methods of chemical analysis.
By Dr. B.K.Sharma, Page no. 661-670.
 Instrumental analysis by William Kemp.
 http://www.intechopen.com/books/gel-
electrophoresisprinciples-and-basics
 Wikipedia .

24. THANK YOU