Electrophoresis of proteins and nucleic acids: II--Techniques and applications.

Sodium dodecyl sulphate polyacrylamide gel electrophoresis This procedure separates poplypeptides according to their size; as it is most commonly used proteins are resolved as a consequence of differences in their rates of migration. SDS/PAGE systems use buffers that contain a detergent, sodium dodecyl sulphate (SDS), which denatures proteins. In a solution of SDS most multi-subunit proteins are denatured and the individual subunits dissociate. Each molecule of SDS carries a negative charge. At neutral pH most proteins become coated uniformly with SDS, which eliminates the intrinsic charge of the protein, so that the total charge of the protein-SDS complex is determined by the SDS. As a consequence, protein-SDS complexes all have a rod-like shape and possess essentially identical charge densities. The electrophoretic mobility of the complex therefore depends on the molecular weight of the protein. During SDS/PAGE protein samples are applied at the cathodal end of a slab of acrylamide gel. Under the influence of the electric field the protein-SDS complexes migrate towards the anode and enter the polyacrylamide gel as a narrow uniform band or zone. Within the gel the migration rate of large proteins is slower than that of small proteins owing to the sieving properties of polyacrylamide (fig 1). To determine the molecular weight of a sample protein standard proteins of known molecular weight are run in parallel with it and their mobility compared.'