A technique for identification of cellular proteins by western immunoblotting and immunoautoradiography of 2-D PAGE.

ABSTRACT

A major effort of our work has been devoted to the identification, using two-dimensional polyacrylamide gel electrophoresis (2-D PAGE), of lymphoid polypeptides that are involved in lymphoid proliferation or differentiation. We have encountered problems during this effort pertaining to the exact localization of a polypeptide(s) in the silver stained gels that is recognized by western immunoblotting or proteins detected by autoradiography. In this paper, we present a method, using India ink stained/immuno-staining replica of 2-D gel nitrocellulose membrane (NCM) or India ink stained coupled with autoradiography in the case of phosphoproteins, which allows us to exactly localize the polypeptide spots detected by these methods in the silver stained 2-D PAGE. This method is expected to popularize and widen the use of 2-D PAGE technology in the investigation of cellular polypeptides.