Purification of a human plasma membrane glycoprotein from human red blood cells by affinity chromatography using a monoclonal antibody.
Using the monoclonal antibody LICR-LON-Fib75.1 coupled to Sepharose as an affinity chromatography column, a membrane glycoprotein with an apparent molecular weight of 18,000 on sodium dodecyl sulfate-polyacrylamide gels has been purified from human red blood cells. The purified protein contained 25% carbohydrate by weight, the predominant sugars being galactose, mannose, and glucosamine. Amino acid analysis indicated that the protein was relatively rich in aspartate, glutamate, valine, and leucine and had a low proline and methionine content. The molecule could be removed from intact red blood cells by trypsin and could be labeled with iodine by lactoperoxidase-catalyzed cell surface iodination of red blood cells. The protein could also be labeled using the lipidsoluble photoactivatable reagent 3-(trifluoromethyl)-3-(m-[125I]iodophenyl) diazirine) and partitioned into the lower phase of the phase-separable detergent Triton X-114. During size-exclusion chromatography in different detergents alterations were observed in the apparent molecular weight of the protein. These results suggest that this Fib75.1-binding protein is an external red blood cell membrane glycoprotein which is capable of binding detergent. Proteins with a similar molecular weight have also been isolated from two human tumor cell lines by immunoprecipitation with this monoclonal antibody.
D000911:Antibodies, Monoclonal; D000936:Antigen-Antibody Complex; D002846:Chromatography, Affinity; D004591:Electrophoresis, Polyacrylamide Gel; D004910:Erythrocyte Membrane / Q000032:analysis*; D006023:Glycoproteins / Q000097:blood* / Q000302:isolation & purification; D006801:Humans; D008565:Membrane Proteins / Q000097:blood* / Q000302:isolation & purification; D008970:Molecular Weight; D014357:Trypsin
D000911:Antibodies, Monoclonal; D000936:Antigen-Antibody Complex; D006023:Glycoproteins; D008565:Membrane Proteins; D014357:Trypsin