Electrophoresis
Dr. Nikhat Siddiqi
1
• A molecule with a net charge will move in an
electric field.
• This phenomenon, termed electrophoresis, offers
a powerful means of separating proteins and
other macromolecules, such as DNA and RNA.
• The velocity of migration (v) of a protein (or any
molecule) in an electric field depends on the
electric field strength (E), the net charge on the
protein (z), and the frictional coefficient (f).
Dr. Nikhat Siddiqi
2
• v = Ez/f
• The electric force Ez driving the charged molecule
toward the oppositely charged electrode is opposed
by the viscous drag fv arising from friction between
the moving molecule and the medium.
• The frictional coefficient f depends on both the mass
and shape of the migrating molecule and the
viscosity (η) of the medium. For a sphere of radius r,
f = 6πηr
Dr. Nikhat Siddiqi
3
Gel Electrophoresis
• Since gels used in biochemistry are chemically
rather unreactive, they interact minimally with
biomolecules during electrophoresis allowing
separation based on physical rather than
chemical differences between sample
components.
Dr. Nikhat Siddiqi
4
• Gel networks may be formed from polymers
which are crosslinked or noncrosslinked.
• Carbohydrate polymeric materials such as
starch were historically used to form gel
networks for the separation of proteins.
Dr. Nikhat Siddiqi
5
• In starch gel electrophoresis of proteins, these effects result in lowresolution electrophoresis with broad bands.
• The most widely-used polysaccharide gel matrix nowadays is that
formed with agarose. This is a polymer composed of a repeating
disaccharide unit called agarobiose which consists of galactose and
3, 6-anhydrogalactose.
• Agarose gives a more uniform degree of porosity than starch and
this may be varied by altering the starting concentration of the
suspension (low concentrations give large pores while high
concentrations give smaller pores).
• This gel has found widespread use especially in the separation of
DNA molecules (although it may also be used in some
electrophoretic procedures involving protein samples such as
immunoelectrophoresis.
Dr. Nikhat Siddiqi
6
• Electrophoretic separations are nearly always carried out in gels (or
on solid supports such as paper) because the gel serves as a
molecular sieve that enhances separation.
• Molecules that are small compared with the pores in the gel readily
move through the gel, whereas molecules much larger than the
pores are almost immobile.
• Intermediate-size molecules move through the gel with various
degrees of facility.
• Electrophoresis is performed in a thin, vertical slab of
polyacrylamide. The direction of flow is from top to bottom.
Polyacrylamide gels, formed by the polymerization of acrylamide
and cross-linked by methylenebisacrylamide, are choice supporting
media for electrophoresis because they are chemically inert and are
readily formed.
Dr. Nikhat Siddiqi
7
Dr. Nikhat Siddiqi
8
High Voltage Paper Electrophoresis
• Low voltage paper electrophoresis is inefficient for
separation of small molecules like amino acids and
nucleotides.
• Filter paper is still employed and found very
satisfactory for use in high voltage electrophoresis.
• The technique is particularly useful for the separation
of low molecular weight substances such as amino
acids, peptides and polypeptides.
• One problem that must be overcome when using high
voltage electrophoresis is the dissipation of the heat
generated by applying appropriate cooling.
Dr. Nikhat Siddiqi
9
• In tank-electrophoresis, during the separation
process, the filter paper is immersed in a
cooled liquid medium such as a chlorinated
hydrocarbon eg carbon tetrachloride or
toluene.
• The technique is particularly useful for
‘fingerprinting proteins.
Dr. Nikhat Siddiqi
10
Dr. Nikhat Siddiqi
11
• In enclosed strip method the paper is not
immersed in the buffer.
• There are cooling plates to cool the paper.
Dr. Nikhat Siddiqi
12
Cellulose acetate strip electrophoresis
• Many biomolecules adsorb to cellulose
(paper) by the hydroxyl group of cellulose.
• Adsorption impedes the movement and
causes tailing of spots.
• This is avoided by using cellulose acetate
membrane where most of the hydroxyl group
have been converted to acetate groups.
Dr. Nikhat Siddiqi
13
Dr. Nikhat Siddiqi
14
Dr. Nikhat Siddiqi
15