Polyacryalamide Gel

Polyacrylamide Gel electrophoresis

Aim: To determine protein molecular weight

Objective: The experiment is performed to determine the molecular weight and most commonly used to separate proteins for the purposes of analysis and purification by comparing it to standard.

Background: In SDS polyacrylamide gel electrophoresis (SDS-PAGE) separations, migration is determined not by intrinsic electric charge of polypeptides but by molecular weight. Sodium dodecylsulphate (SDS) is an anionic detergent that denatures proteins by wrapping the hydrophobic tail around the polypeptide backbone. For almost all proteins, SDS binds at a ratio of approximately 1.4 g SDS per gram of protein, thus conferring a net negative charge to the polypeptide in proportion to its length.

The SDS also disrupts hydrogen bonds, blocks hydrophobic interactions, and substantially unfolds the protein molecules, minimizing differences in molecular form by eliminating the tertiary and secondary structures. The proteins can be totally unfolded when a reducing agent such as dithiothreitol (DTT) is employed. DTT cleaves any disulphide bonds between cysteine residues. The SDS-denatured and reduced polypeptides are flexible rods with uniform negative charge per unit length. Thus, because molecular weight is essentially a linear function of peptide chain length, in sieving gels the proteins separate by molecular weight.

There are two types of buffer systems used in protein gel electrophoresis: continuous and discontinuous. A continuous system uses only one buffer for the tanks and the gel. In a discontinuous system, first developed by Ornstein (1964) and Davis (1964), a nonrestrictive large-pore gel called a stacking gel is layered on top of a separating (resolving) gel. The two gel layers are each made with a different buffer, and the tank buffers differ from the gel buffers.

In a discontinuous system, protein mobility—a quantitative measure of the migration rate of a charged species in an...