Transcript lecture-1x
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