Polyacrylamide Gel Affinity Electrophoresis for Separation of Enzyme Isoforms.

Affinity electrophoresis of differently glycosylated isoforms of enzymes using lectin as affinity ligand has been reported on support media such as cellulose acetate membrane (CAM) or agarose gel. We report a method for affinity electrophoresis in polyacrylamide gel (PAG) using wheat germ agglutinin (WGA). WGA is added to acrylamide-Bis mixture and incubated for 10 minutes at room temperature. This causes WGA to react covalently with acrylamide and Bis. Polymerization is initiated with addition of N,N,N,N-tetramethylethylene diamine (TEMED) and ammonium persulphate to give polyacrylamide gel with immobilized lectin. This gel has been found to effectively separate differently glycosylated isoforms of alkaline phosphatase (ALP). Concanavalin - A, similarly immobilized, did not give effective separation of ALP isoforms. The immobilization of lectin on polyacrylamide as support media requires less amount of lectin in comparison to CAM and agarose. Additional advantage of affinity electrophoresis on PAG is separation of biomolecules according to size.