Large-scale purification of murine I-Ak and I-Ek antigens and characterization of the purified proteins.
Detailed analysis of the role of the structural characteristics of these molecules will require isolation of relatively large amounts of these antigens in serologically active form. We have purified murine Ia antigens on a large scale by affinity chromatography using monoclonal antibodies coupled to Sepharose 4B. Both I-Ak and I-Ek were isolated by sequential passage of cell lysate over columns prepared using specific monoclonal antibodies. Elution of the bound antigens required high pH (11-12) but, nonetheless, the purified material was 50-75% serologically active. Using LPS-stimulated spleen cells or B-lymphocyte tumor cells as starting material, 0.5 mg of each antigen can be readily purified. Based on antigen yields, it can be estimated that normal B-cells have about the same surface density of Class I and Class II MHC antigens. LPS blasts, in contrast, have normal levels of Class I antigen but 3-5 times higher levels of Class II antigens. We have now purified I-Ak and I-Ek from a number of different cell sources and have noted differences in both the mol. wts of the alpha- and beta-chains and in their apparent associations with cytoskeletal components. Proteins having the same apparent mol. wts as actin and myosin co-purify with both I-Ak and I-Ek antigens from various sources. These proteins do not co-purify with H-2K and D molecules obtained by similar methods, suggesting that Ia antigens may specifically interact with cytoskeletal elements.
D000199:Actins; D000818:Animals; D000911:Antibodies, Monoclonal / Q000276:immunology; D002460:Cell Line; D002846:Chromatography, Affinity; D002847:Chromatography, Agarose; D004591:Electrophoresis, Polyacrylamide Gel; D000949:Histocompatibility Antigens Class II / Q000302:isolation & purification*; D008214:Lymphocytes / Q000276:immunology; D008223:Lymphoma / Q000276:immunology; D051379:Mice; D008815:Mice, Inbred Strains; D008970:Molecular Weight; D009218:Myosins; D010455:Peptides
D000199:Actins; D000911:Antibodies, Monoclonal; D000949:Histocompatibility Antigens Class II; D010455:Peptides; D009218:Myosins