The purification of murine histocompatibility antigens (H-2b) from RBL-5 tumor cells using detergents.
A detergent-solubilized form of H-2b (dH-2b) has been purified 1500-fold from RBL-5 tumor cells. The purification was accomplished by deoxycholate solubilization of purified plasma membranes, gel filtration, Lens culinaris lectin affinity chromatography, and affinity chromatography on a sheep anti-dH-2b immunoadsorbent. Both alloantigen and beta 2-microglobulin were monitored by radioimmunoassay during purification. The final product was judged to be greater than 90% pure by the following criteria: 1) sodium dodecyl sulfate-polyacrylamide electrophoresis which showed the expected 2-component structure of histocompatibility antigens, i.e. a heavy chain and beta 2-microglobulin; 2) amino acid composition which was comparable to the known compositions of other H-2 and HLA molecules; 3) NH2-terminal sequencing which gave a unique sequence for the heavy chain, and the reported sequence for beta 2-microglobulin; and 4) immunoprecipitation of the bulk of the preparation by appropriate alloantisera.
D000595:Amino Acid Sequence; D000596:Amino Acids / Q000032:analysis; D000818:Animals; D002462:Cell Membrane / Q000276:immunology; D002850:Chromatography, Gel; D003840:Deoxycholic Acid; D006649:Histocompatibility Antigens / Q000302:isolation & purification*; D007942:Leukemia, Experimental / Q000276:immunology*; D008099:Liver / Q000276:immunology; D051379:Mice; D008970:Molecular Weight; D011863:Radioimmunoassay
D000596:Amino Acids; D006649:Histocompatibility Antigens; D003840:Deoxycholic Acid