Immunoblotting and dot blotting

Abstract

A variety of methods has been available for many years for the analytical separation of mixtures of proteins into their component parts by electrophoresis in a gel, usually agarose or polyacrylamide. Popular techniques are: zonal electrophoresis in agarose gel or on cellulose acetate membranes; discontinuous electrophoresis in polyacrylamide gel (PAGE); SDS-polyacrylamide gel electrophoresis (SDS-PAGE); isoelectric focusing (IEF); and two-dimensional polyacrylamide gel electrophoresis (2-D PAGE), which is capable of resolving complex mixtures of proteins containing hundreds or even thousands of components. All of these methods require some means of identifying particular proteins of interest. In some cases it is possible to do this simply on the basis of mobility, molecular weight (MW) (SDS-PAGE) or by using selective stains, e.g., for enzyme activity. These methods of identification are severely limited in applicability and, for this reason, antibodies have been used as highly specific probes for electrophoretically separated proteins, ever