Casting of the gel: The acrylamide solution (a mixture of monomeric acrylamide and a bifunctional crosslinker bisacrylamide ) is mixed with the TEMED and APS and poured in between the glass plate fitted into the gel caster. What is the mechanism of acrylamide polymerization? Ammoinum persulfate in the presence of TEMED forms oxygen free radicals and induces the polymerization of acryalide monomer to form a linear polymer (Figure 13.4). These linear monomers are interconnected by the cross linking with bis-acrylamide monomer to form a 3-D mesh with pores. The size of pore is controlled by the concentration of acrylamide and amount of bis-acrylamide in the gel. In a vertical gel electrophoresis system, we cast two types of gels, stacking gel and resolving gel. First the resolving gel solution is prepared and poured into the gel cassette for polymerization. A thin layer of organisc solvent (such as butanol or isoproponal) is layered to stop the entry of oxygen (oxygen neutralizes the free radical and slow down the polymerization) and make the top layer smooth. After polymerization of the resolving gel, a stacking gel is poured and comb is fitted into the gel for construction of different lanes for the samples (Figure 13.5).

Figure 13.4 : Mechanism of acylamide polymerization.

Figure 13.5 : Different steps in performace of vertical gel electrophoresis to resolve sample.