Monoclonal antibody immunoprecipitation of cell membrane glycoproteins.
Procedural modifications facilitating the immunoprecipitation of cell surface-associated glycoproteins by monoclonal antibodies are presented. The use of complexes of antibodies coupled to protein A-Sepharose in place of antibodies directly coupled to Sepharose, and the inclusion of ATP and salt in the lysis buffer, is shown to markedly reduce the nonspecific binding of aggregated cytoskeletal proteins. These modifications result in low backgrounds while the specific membrane-associated proteins are still quantitatively immunoprecipitated.
Howard Hughes Medical Institute, University of California, San Francisco 94143.
G A
GA
Peltz
B
B
Gallis
B M
BM
Peterlin
D000255:Adenosine Triphosphate; D000911:Antibodies, Monoclonal; D002460:Cell Line; D002462:Cell Membrane / Q000032:analysis; D011232:Chemical Precipitation; D006023:Glycoproteins / Q000276:immunology / Q000302:isolation & purification*; D006801:Humans; D008565:Membrane Proteins / Q000276:immunology / Q000302:isolation & purification*; D011961:Receptors, Fc / Q000276:immunology / Q000302:isolation & purification; D012685:Sepharose / Q000031:analogs & derivatives; D013205:Staphylococcal Protein A
D000255:Adenosine Triphosphate; D000911:Antibodies, Monoclonal; D006023:Glycoproteins; D008565:Membrane Proteins; D011961:Receptors, Fc; D012685:Sepharose; C042030:Staphylococcal protein A-sepharose; D013205:Staphylococcal Protein A