Gel electrophoretic separation of polypeptides can be achieved on the basis of size and charge (gel electrophoresis under non-dissociating conditions), size alone (SDS-PAGE) or charge alone (isoelectric focusing, IEF). Irrespective of the method chosen, there is a real possibility that two or more polypeptides may co-migrate as a single band, particularly if the original sample is a complex mixture of proteins. This co-migration of proteins will mask the complexity of a protein mixture and so can lead to incorrect conclusions as to the purity of protein samples.

Two-dimensional gel electrophoretic methods have been devised which separate proteins on the basis of charge in one dimension followed by separation on the basis of size in a second dimension. The first-dimensional separation on the basis of charge is carried out by electrophoresis of the protein sample in a rod polyacrylamide gel, usually by IEF. After electrophoresis, this rod gel is arranged to lie horizontally against the top edge of the stacking gel of a slab gel prepared for SDS-PAGE. During this second dimensional electrophoresis, the polypeptides separated by charge in the rod gel during the first stage electrophoresis now migrate into the slab gel where they separate on the basis of size. Either uniform-concentration polyacrylamide slab gels or concentration-gradient slab gels may be used for the second dimensional SDS-PAGE step. The separated proteins appear as spots after staining. If one measures the pH gradient in the first-dimensional rod gel and also co-electrophoreses polypeptides of known molecular weight down the side of the slab gel during the second-dimensional SDS-PAGE step, one can estimate both the isoelectric point and the molecular weight of any sample polypeptide of interest simply by noting its horizontal position and vertical position respectively.

Because of the two-stage separation involved, two-dimensional polyacrylamide gel electrophoresis is the method of highest resolution of all the protein electrophoretic methods in current use. As such it is an extremely valuable test of purity of a protein sample. Thus, although SDS-PAGE or IEF may well be used to monitor the purity of a protein sample at several stages of a purification scheme, two-dimensional gel electrophoresis should be used as the final test when the protein of interest appears to be pure. Note, however, that there is no guarantee that a single spot after two-dimensional gel electrophoresis is only a single polypeptide; the use of two-dimensional gel electrophoresis simply minimises the possibility of co-migration of polypeptides. Indeed there are occasions when two or more polypeptides may well co-migrate, not because they have identical size and charge, but because they are interacting with each other and so migrate as an aggregate rather than individually. For example, proteins isolated from membranes may form very stable aggregates that resist the normal conditions of dissociation employed by IEF and SDS-PAGE. In these cases one may need to include additional reagents during sample preparation and electrophoresis, such as urea, to ensure complete dissociation of the proteins.