Hapten-mediated immunopurification of membrane proteins labeled with fluorescein derivatives.
The behavior of cell surface components labeled with fluorochromes can be studied by fluorescence microscopy and spectroscopy; further structural analyses would be facilitated by purification of the labeled components. We have developed a protocol for identifying the targets for labeling with fluorescein derivatives, by using 125I- diiodofluorescein isothiocyanate ( 125IFC ) and for isolating the labeled components with anti-IFC immunoadsorbents. Anti-IFC antibodies obtained from rabbits immunized with IFC-hemocyanin were purified by affinity chromatography and coupled to CNBr-activated Sepharose 4B. The anti-IFC immunoadsorbents could then be used to isolate the entire set of 125IFC -proteins from crude detergent extracts of labeled sea urchin sperm, with a 70% yield and a purification of more than 250 fold. Nonspecific binding of unlabeled proteins to the immunoadsorbent was insignificant. When the immunoadsorbent IFC-protein complex was used directly as an immunogen, antibodies were obtained that reacted with the underivatized proteins that were targets for IFC labeling, as indicated by immunoblotting after gel electrophoresis. The antibodies also reacted with the surface of unlabeled sperm as shown by immunofluorescence. Thus, by treating the IFC-sperm proteins as a class, we obtained antibodies that recognized the unlabeled proteins in situ or in cell extracts. This approach should be generally useful in obtaining reagents directed against specific cell surface components.
D000818:Animals; D000918:Antibody Specificity; D002846:Chromatography, Affinity; D016650:Fluorescein-5-isothiocyanate; D005452:Fluoresceins; D005455:Fluorescent Antibody Technique; D006241:Haptens / Q000276:immunology*; D005779:Immunodiffusion; D007163:Immunosorbent Techniques; D017879:Isothiocyanates; D008297:Male; D008565:Membrane Proteins / Q000302:isolation & purification*; D011817:Rabbits; D012617:Sea Urchins; D013094:Spermatozoa / Q000166:cytology; D013861:Thiocyanates
D005452:Fluoresceins; D006241:Haptens; D017879:Isothiocyanates; D008565:Membrane Proteins; D013861:Thiocyanates; C016393:diiodofluorescein isothiocyanate; D016650:Fluorescein-5-isothiocyanate