Biochemical Techniques and Instrumentation

5.3.2.2.Gel electrophoresis

5.2.3.2. Gel electrophoresis

Proteins are separated using Sodium dodecyl sulphate-Polyacrylamide (SDS-PAGE) gel electrophoresis based on their molecular weight and electric charge. SDS-PAGE maintains polypeptides in a denatured state. SDS removes the secondary and the tertiary structure of the protein by disrupting the disulfide bonds [S-S] to sulfhydryl groups [SH and SH]. It also covers the denatured proteins with negative charge and move them to the positively charged electrode through the acrylamide mesh of the gel. Smaller proteins migrate faster through the mesh and the proteins are thus separated according to size, usually measured in kilodaltons, kDa. One lane is usually reserved for a marker or ladder, a commercially available mixture of proteins having defined molecular weights. It is also possible to use a two-dimensional (2-D) gel which spreads the proteins from a single sample in two dimensions. Proteins are separated according to isoelectric point in the first dimension, and according to their molecular weight in the second dimension.