Rapid quantitative separation of the major glycoproteins (PAS 1, 2, and 3) from other human red cell membrane proteins in a nondenaturing medium by affinity chromatography

Several methods were tested for separating the major glycoproteins from other proteins extracted from human red cell membranes by the nondenaturing detergent, Triton X-100. Separation could be achieved by isoelectric focusing, but the intrinsic proteins (predominantly band 3) become irreversibly precipitated. Dithiol-containing resin (Thiol-Sepharose, Pharmacia) was found to be capable of removing the glycoproteins [Kahlenberg, A. (1976) Anal. Biochem. 74, 337–342], but the procedure is relatively slow. Rapid and quantitative separation was achieved with an organomercurial gel (Affi-Gel 501, Bio-Rad). The major glycoproteins were not retarded, but all of the other membrane proteins were retained in the gel. They could be subsequently eluted with solutions containing cysteine.