Buffer and reagent for electrophoresis- The different buffer and reagents with their purpose for vertical gel electrophoresis is as follows-

1. N, N, N', N'-tetramethylethylenediamine (TEMED)-it catalyzes the acrylamide polymerization.

2. Ammonium persulfate (APS)-it is an initiator for the acrylamide polymerization.

3. Tris-HCl- it is the component of running and gel casting buffer.

4. Glycine- it is the component of running buffer.

5. Bromophenol blue- it is the tracking dye to monitor the progress of gel electrophoresis.

6. Coomassie brilliant blue R250-it is used to stain the polyacrylamide gel.

7. Sodium dodecyl sulphate-it is used to denature and provde negative charge to the protein.

8. Acrylamide- monomeric unit used to prepare the gel.  

9. Bis-acrylamide- cross linker for polymerization of acrylamide monomer to form gel.  

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. Ammoinum persupfate in the presence of TEMED forms oxygen free radicals and induces the polymerization of acryalide monomer to form a linear polymer (Figure 35.3). 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 35.4).