Isolation of the I-Ak core complex and its associated gamma-polypeptide by affinity chromatography on monoclonal immunoadsorbent 10-2.16 sepharose CL-4B.
Spleen cell-derived I-Ak antigens have previously been shown by us to be comprised of four noncovalently associated polypeptide chains (alpha, gamma, beta 1, beta 2). Here we report that pretreatment of the detergent solubilized four-chain structure with chaotropic ions yielded an alloantigenically reactive core complex (alpha/beta 1, beta 2) and a dissociated polypeptide gamma-chain which was not recognized by monoclonal antibody 10-2.16 (anti-I-Ak). The lack of alloantigenic determinants on gamma and the observation that gamma did not reassociate with alpha/beta 1, beta 2 upon removal of the chaotropic reagent suggested a rapid purification procedure for both moieties. A pure preparation of the serologically intact core complex was achieved by repeated affinity chromatography on hybridoma immunoadsorbent 10-2.16 Sepharose CL-4B. Concurrently its pertinent gamma-polypeptide was obtained in a highly enriched form and was purified to homogeneity by an additional gel electrophoretic step.
D000818:Animals; D000911:Antibodies, Monoclonal / Q000276:immunology; D002846:Chromatography, Affinity; D004591:Electrophoresis, Polyacrylamide Gel; D000939:Epitopes / Q000032:analysis; D000949:Histocompatibility Antigens Class II / Q000276:immunology / Q000378:metabolism*; D046911:Macromolecular Substances; D051379:Mice; D008809:Mice, Inbred C3H; D010455:Peptides / Q000302:isolation & purification; D012685:Sepharose / Q000031:analogs & derivatives / Q000276:immunology
D000911:Antibodies, Monoclonal; D000939:Epitopes; D000949:Histocompatibility Antigens Class II; D046911:Macromolecular Substances; D010455:Peptides; C035771:Sepharose CL 4B; D012685:Sepharose