Chapter 32 : Protein Electrophoresis

Abstract:

In this activity, students will perform agarose gel electrophoresis with amylase samples. The goal of this student activity is to separate the proteins in amylase-containing samples by electrophoresis so that students can see different protein bands and to identify amylase bands through their starch-hydrolyzing activity. Electrophoresis of proteins is different from electrophoresis of DNA in a few important respects. A mixture of proteins applied to an electrophoretic gel will likely contain some of each. Proteins assume different shapes, so two proteins of the same molecular weight and charge might migrate differently in a gel. To make proteins migrate more uniformly, they are often treated with the detergent sodium dodecyl sulfate (SDS). Proteins in SDS buffer migrate toward the positive electrode in an electric field, and the distances they migrate are largely proportional to their molecular weights. The gels in this activity are made with fine-sieving agarose, which improves the resolution of protein bands. Starch is also added to the gel. As the amylase activity is reconstituted, the enzyme degrades the starch in its vicinity, creating an area of clearing in the gel. Students can compare the stained half of the gel to the half with amylase activity.