A TECHNIQUE FOR IDENTIFICATION OF CELLULAR PROTEINS BY WESTERN IMMUNOBLOTTING AND IMMUNOAUTORADIOGRAPHY OF 2-D PAGE

A major effort of our work has been devoted to the identification, usi ng two-dimensional polyacrylamide gel electrophoresis (2-D PAGE), of l ymphoid polypeptides that are involved in lymphoid proliferation or di fferentiation. We have encountered problems during this effort pertain ing to the exact localization of a polypeptide(s) in the silver staine d gels that is recognized by western immunoblotting or proteins detect ed by autoradiography. In this paper, we present a method, using India ink stained/immuno-staining replica of 2-D gel nitrocellulose membran e (NCM) or India ink stained coupled with autoradiography in the case of phosphoproteins, which allows us to exactly localize the polypeptid e 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 techno logy in the investigation of cellular polypeptides.