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 electric field — is intermediate between the mobility of the buffer ion of the same charge (usually negative) in the stacking gel (leading ion) and the mobility of the buffer ion in the upper tank (trailing ion). When electrophoresis is started, the ions and the proteins begin migrating into the stacking gel. The proteins concentrate in a very thin zone, called the stack, between the leading ion and the trailing ion. The proteins continue to migrate in the stack until they reach the separating gel. At that point, due to a pH or an ion change, the proteins become the trailing ion and "unstack" as they separate on the gel. Although a continuous system is slightly easier to set up than a discontinuous system and tends to have fewer sample precipitation and aggregation problems, much greater resolution can be obtained with a discontinuous system. Only minimal concentration of the sample takes place with continuous gels, and proteins form zones nearly as broad as the height of the original samples in the sample wells, resulting in much lower resolution.

Denaturing gel electrophoresis can resolve complex protein mixtures into hundreds of bands on a gel. The position of a protein along the separation lane gives a good approximation of its size, and, after staining, the band intensity is a rough indicator of the amount present in the sample. This simultaneous ability to estimate size and amount of a protein is useful in various applications: estimating purity, level of gene expression, immunoblotting, preparing for protein sequencing, and generating antibodies.

The discontinuous Laemmli system (Laemmli, 1970), a denaturing modification of Ornstein (1964) and Davis (1964), is the most widely used system for research protein electrophoresis today. The resolution in a Laemmli gel is excellent because the treated peptides are concentrated in a stacking zone before entering the separating gel. Other buffer systems can be used, for example the Tris ™ -tricine system of Schägger and von Jagow (1987) for resolution of polypeptides in the size range below Mr 10 000. For a detailed discussion and general reference on denaturing gel electrophoresis, consult the references listed at the end of this section.