Solubilisation effect of Nonidet P-40, triton X-100 and CHAPS in the detection of MHC-like glycoproteins.
We have analysed the differential solubilisation effect of three detergents on cell-membrane histocompatibility glycoproteins. Two nonionic detergents (Nonidet P-40 and Triton X-100) which are extensively used in the extraction of MHC proteins and a zwitterionic detergent (CHAPS) which is sulphobetaine derivative of cholic acid were used. An AKR (H-2k) derived spontaneous leukaemic cell line--424--was used as the experimental model. In this tumour cell line a class I-like antigen is expressed but not directly detected by cell-binding radioimmunoassay or immunoprecipitation from NP-40 or Triton X-100 solubilised glycoproteins. However, 46 kDa and 12 kDa bands consistent with the classical H-2 class I pattern were seen by SDS-PAGE after immunoprecipitation with the 34.5.8 anti-H-2Dd MoAb using CHAPS solubilised 424 glycoproteins. The H-2Dd-reactive molecule appears to be associated with at least one of the syngeneic class I specificities (H-2Kk, H-2Dk) and not accessible to react with the specific anti H-2Dd MoAb. The detergents NP-40 and Triton X-100 appear to be less efficient than CHAPS in breaking protein-protein interactions. This property of CHAPS permitted the adequate solubilisation of the novel antigen and its direct detection. The results of this study suggest that the alternative use of a non-denaturing zwitterionic detergent may contribute to the detection and characterisation of MHC-related, membrane-bound proteins of tumours and normal cells.
Labeta
M O
MO
Department of Immunology, London Hospital Medical College, U.K.
Fernandez
N
N
Festenstein
H
H
D000818:Animals; D000911:Antibodies, Monoclonal; D002460:Cell Line; D002793:Cholic Acids; D003902:Detergents; D004591:Electrophoresis, Polyacrylamide Gel; D006183:H-2 Antigens / Q000302:isolation & purification*; D008562:Membrane Glycoproteins / Q000302:isolation & purification*; D051379:Mice; D008806:Mice, Inbred AKR; D017830:Octoxynol; D011092:Polyethylene Glycols; D011233:Precipitin Tests; D012967:Sodium Dodecyl Sulfate; D012995:Solubility; D013501:Surface-Active Agents
D000911:Antibodies, Monoclonal; D002793:Cholic Acids; D003902:Detergents; D006183:H-2 Antigens; D008562:Membrane Glycoproteins; D013501:Surface-Active Agents; D012967:Sodium Dodecyl Sulfate; D011092:Polyethylene Glycols; D017830:Octoxynol; C010615:Nonidet P-40; C028213:3-((3-cholamidopropyl)dimethylammonium)-1-propanesulfonate