Resolving Proteins for Immunoblotting by Gel Electrophoresis

Abstract

This protocol describes Tris/glycine SDS–polyacrylamide gel electrophoresis, also known as SDS discontinuous gel electrophoresis or the Laemmli electrophoresis system. The gel-casting unit is assembled and tested to make sure that there are no leaks. Ammonium persulfate and tetramethylethylenediamine are added to the separating monomer solution, and the bottom (separating) gel is poured and allowed to polymerize. The top (stacking) gel is poured, and the comb is inserted to make the wells. The stacking gel is allowed to polymerize and the comb is then removed. The gel cassette is connected to the buffer chambers and the samples are loaded into the wells. When the electric current is applied, the proteins migrate through the gel with a rate that is roughly proportional to the length of their polypeptide chains. The electrophoresis is stopped when the loading dye reaches the bottom of the separating gel. The gel cassette is disassembled, and the proteins are ready for transfer from the gel onto the membrane.