Preparation and characterization of monoclonal antibodies to several frog rod outer segment proteins

• Published in: The Journal of general physiology Vol. 84; no. 2; pp. 251 - 263
• Main Authors: WITT, P. L, HAMM, H. E, BOWNDS, M. D
• Format: Journal Article
• Language: English
• Published: New York, NY Rockefeller University Press 01.08.1984; The Rockefeller University Press
• Subjects: Animals; Antibodies, Monoclonal - isolation & purification; Antibody Affinity; Binding Sites, Antibody; Biological and medical sciences; Cell physiology; Cytoplasm - immunology; Eye Proteins - immunology; Fluorescent Antibody Technique; Fundamental and applied biological sciences. Psychology; Heterotrimeric GTP-Binding Proteins; Light; Molecular and cellular biology; monoclonal antibodies; Photoreceptor Cells - immunology; Photoreceptor Cells - physiology; proteins; Rana; Ranidae; Rhodopsin - immunology; Rod Cell Outer Segment - immunology; Rod Cell Outer Segment - ultrastructure; rod outer segment membranes; Vision, photoreception; Proteins; Vertebrata; Outer segment; Rod; Preparation; Amphibia; Retina; Monoclonal antibody
• ISSN: 0022-1295; 1540-7748
• DOI: 10.1085/jgp.84.2.251

Monoclonal antibodies to proteins important in phototransduction in the frog rod outer segment have been obtained. These include 6 different antibodies to rhodopsin, 50 to a guanine nucleotide binding protein (G-protein; 40,000 daltons), and 2 to cytoplasmic proteins. The antigens used were Percoll-purified rod outer segments, a rod outer segment soluble protein fraction, or a soluble plus peripheral membrane protein fraction. Antibodies were assayed by solid phase assay using a fluorogenic detection system. Proteins to which antibodies bound were assayed on Western blots, and the sensitivities of three different detection systems were compared. Most antibodies bound to only one rod outer segment protein band on Western blots. Immunofluorescence microscopy demonstrated binding of both anti-rhodopsin and anti-G-protein to isolated frog rod outer segments. Antibodies were purified from either culture supernatants or ascites fluid on protein A affinity columns. Two purified anti-G-protein antibodies have binding affinities to 125I-labeled G-protein of less than 10(-6) M-1. Of 11 antibodies to frog or bovine G-protein tested in solid phase and Western blot assays, all bind to the alpha rather than the beta or gamma subunits. Procedures developed here are being used in preparing other antibodies that affect reactions in the phototransduction pathway.