Isolation of cell surface proteins by hybridization

Abstract

Human erythrocytes were treated with the diazonium salt of oligodeoxythymidylic acid 5′- p-aminophenylphosphate, a reagent that does not penetrate the plasma membrane. Ghosts were isolated, and the oligomers, covalently linked at their 5′ ends to the outer surface of the membrane, were extended by treatment with terminal deoxynucleotidyl transferase in the presence of deoxythymidine triphosphate. The membranes were dissolved in sodium dodecyl sulfate, and complexes containing cell surface components were isolated by hybridization to polyriboadenylic acid-agarose. The cell surface components were regenerated by treatment with nuclease P 1 in the presence of Triton X100. Sodium dodecyl sulfate/polyacrylamide gels of the regenerated material showed bands III, PAS-1, PAS-2, and PAS-3, i.e. the major proteins known to be accessible at the outer surface of the human erythrocyte. The method should be useful for the isolation of surface components in other cell types.