Membrane protein analysis by two-dimensional immunoelectrophoresis

Abstract

Water-soluble membrane proteins may be analyzed by a new, rapid technique that combines electrophoresis on high-resolution sodium dodecyl sulfate (SDS) polyacrylamide gels and immunoelectrophoresis. After separation in the first dimension by electrophoresis in SDS, the proteins are subjected to a second electrophoresis at right angles through a two-layered buffered agarose gel. They first pass through a layer containing Lubrol PX which forms complexes with free SDS and then into an antiserum layer where antigen-antibody precipitates form. Precipitin arcs appear at positions corresponding to the antigens separated in the first dimension. The effectiveness of the technique was demonstrated with frog and cattle opsins, human erythrocyte membrane proteins, and their rabbit antiserums and for several water soluble proteins. By this method two fundamental parameters, molecular weight and antigenicity, may be readily used for analysis of membrane proteins.