Purification and photoaffinity labeling of the I-Ak histocompatibility molecule.
Détails
ID Serval
serval:BIB_92FB738650C5
Type
Article: article d'un périodique ou d'un magazine.
Collection
Publications
Institution
Titre
Purification and photoaffinity labeling of the I-Ak histocompatibility molecule.
Périodique
Journal of Immunological Methods
ISSN
0022-1759 (Print)
ISSN-L
0022-1759
Statut éditorial
Publié
Date de publication
1990
Peer-reviewed
Oui
Volume
135
Numéro
1-2
Pages
233-245
Langue
anglais
Notes
Publication types: Journal Article ; Research Support, Non-U.S. Gov't ; Research Support, U.S. Gov't, P.H.S.
Publication Status: ppublish
Publication Status: ppublish
Résumé
Photoaffinity labeling was used to evaluate optimal conditions for purification of I-A k histocompatibility molecules in functionally active form. We assessed the biological activity of I-A k primarily by its binding of the hen egg-white lysozyme (HEL) peptide from residues 46-61. [125I]iodo,4-azidosalicyloly(HEL)46-61 (IASA-46-61)-labeled I-A k on B cell hybridoma membranes and their detergent solubilisates, at the alpha chain. Following extensive detergent dialysis, the intensity of this labeling remained unchanged in the case of MEGA 8 and MEGA 9 detergents, but decreased in the case of deoxycholate and n-octylglucoside. Conditions for affinity purifications were assessed on one hand by determining the dissociation conditions of I-A k from various monoclonal antibodies and by determining the denaturation of I-A k under these conditions. Effective dissociation in the absence of detectable denaturation was observed for 10.3.6.2 and 40.LH monoclonal antibody at pH 3.5 and to a lesser extent at low concentrations of ammonium thiocyanate and guanidine thiocyanate at neutral pH. I-A k purified from cell membranes using MEGA 8 and MEGA 9 detergent mixtures and acid elution from 10.3.6.2 Sepharose was efficiently labeled by IASA-46-61. Thus I-A k was active in antigen presentation to a T cell hybridoma when reconstituted in planar membranes. In contrast to I-A k on cell membranes, purified I-A k in detergent showed extensive labeling of the beta chain. The overall labeling intensity and the extent of beta chain labeling substantially changed upon addition of certain lysophosphatides.
Mots-clé
Affinity Labels, Antibodies, Monoclonal/immunology, Azides, Cell Membrane/chemistry, Chromatography, Affinity/methods, Deoxycholic Acid/pharmacology, Detergents, Electrophoresis, Polyacrylamide Gel, Fatty Acids, Glucosamine/analogs & derivatives, Glucosides/pharmacology, Histocompatibility Antigens Class II/isolation & purification, Hydrogen-Ion Concentration, Muramidase, Peptide Fragments, Sorbitol/analogs & derivatives, Tumor Cells, Cultured
Pubmed
Web of science
Création de la notice
28/01/2008 12:19
Dernière modification de la notice
20/08/2019 15:55