PMCCPMCCPMCC

Search tips
Search criteria 

Advanced

 
Logo of jgenphysiolHomeThe Rockefeller University PressEditorsContactInstructions for AuthorsThis issue
 
J Gen Physiol. 1984 August 1; 84(2): 251–263.
PMCID: PMC2228734

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

Abstract

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.

Full Text

The Full Text of this article is available as a PDF (1.0M).

Selected References

These references are in PubMed. This may not be the complete list of references from this article.
  • Barnstable CJ. Monoclonal antibodies which recognize different cell types in the rat retina. Nature. 1980 Jul 17;286(5770):231–235. [PubMed]
  • Clark R, Lane DP, Tjian R. Use of monoclonal antibodies as probes of simian virus 40 T antigen ATPase activity. J Biol Chem. 1981 Nov 25;256(22):11854–11858. [PubMed]
  • Clark SP, Molday RS. Orientation of membrane glycoproteins in sealed rod outer segment disks. Biochemistry. 1979 Dec 25;18(26):5868–5873. [PubMed]
  • Dewey MM, Davis PK, Blasie JK, Barr L. Localization of rhodopsin antibody in the retina of the frog. J Mol Biol. 1969 Feb 14;39(3):395–405. [PubMed]
  • Ey PL, Prowse SJ, Jenkin CR. Isolation of pure IgG1, IgG2a and IgG2b immunoglobulins from mouse serum using protein A-sepharose. Immunochemistry. 1978 Jul;15(7):429–436. [PubMed]
  • Frackelton AR, Jr, Rotman B. Functional diversity of antibodies elicited by bacterial beta-D-galactosidase. Monoclonal activating, inactivating, protecting, and null antibodies to normal enzyme. J Biol Chem. 1980 Jun 10;255(11):5286–5290. [PubMed]
  • Fung BK, Hurley JB, Stryer L. Flow of information in the light-triggered cyclic nucleotide cascade of vision. Proc Natl Acad Sci U S A. 1981 Jan;78(1):152–156. [PubMed]
  • Gabay J, Schwartz M. Monoclonal antibody as a probe for structure and function of an Escherichia coli outer membrane protein. J Biol Chem. 1982 Jun 25;257(12):6627–6630. [PubMed]
  • Hamm HE, Bownds MD. A monoclonal antibody to guanine nucleotide binding protein inhibits the light-activated cyclic GMP pathway in frog rod outer segments. J Gen Physiol. 1984 Aug;84(2):265–280. [PMC free article] [PubMed]
  • Hermolin J, Karell MA, Hamm HE, Bownds MD. Calcium and cyclic GMP regulation of light-sensitive protein phosphorylation in frog photoreceptor membranes. J Gen Physiol. 1982 Apr;79(4):633–655. [PMC free article] [PubMed]
  • Hubbell WL, Bownds MD. Visual transduction in vertebrate photoreceptors. Annu Rev Neurosci. 1979;2:17–34. [PubMed]
  • Jan LY, Revel JP. Ultrastructural localization of rhodopsin in the vertebrate retina. J Cell Biol. 1974 Aug;62(2):257–273. [PMC free article] [PubMed]
  • Jay G, Khoury G, DeLeo AB, Dippold WG, Old LJ. p53 transformation-related protein: detection of an associated phosphotransferase activity. Proc Natl Acad Sci U S A. 1981 May;78(5):2932–2936. [PubMed]
  • Kiehart DP, Mabuchi I, Inoué S. Evidence that myosin does not contribute to force production in chromosome movement. J Cell Biol. 1982 Jul;94(1):165–178. [PMC free article] [PubMed]
  • Kirschenbaum DM. Molar absorptivity and A1 percent 1cm values for proteins at selected wavelengths of the ultraviolet and visible regions. IX. Anal Biochem. 1973 Nov;56(1):237–263. [PubMed]
  • Köhler G, Milstein C. Continuous cultures of fused cells secreting antibody of predefined specificity. Nature. 1975 Aug 7;256(5517):495–497. [PubMed]
  • Kühn H. Light-regulated binding of rhodopsin kinase and other proteins to cattle photoreceptor membranes. Biochemistry. 1978 Oct 17;17(21):4389–4395. [PubMed]
  • Kühn H. Light- and GTP-regulated interaction of GTPase and other proteins with bovine photoreceptor membranes. Nature. 1980 Feb 7;283(5747):587–589. [PubMed]
  • Lee RH, Brown BM, Lolley RN. Protein kinases of retinal rod outer segments: identification and partial characterization of cyclic nucleotide dependent protein kinase and rhodopsin kinase. Biochemistry. 1981 Dec 22;20(26):7532–7538. [PubMed]
  • MacKenzie D, Molday RS. Organization of rhodopsin and a high molecular weight glycoprotein in rod photoreceptor disc membranes using monoclonal antibodies. J Biol Chem. 1982 Jun 25;257(12):7100–7105. [PubMed]
  • Mancillas JR, McGinty JF, Selverston AI, Karten H, Bloom FE. Immunocytochemical localization of enkephalin and substance P in retina and eyestalk neurones of lobster. Nature. 1981 Oct 15;293(5833):576–578. [PubMed]
  • Markwell MA, Fox CF. Surface-specific iodination of membrane proteins of viruses and eucaryotic cells using 1,3,4,6-tetrachloro-3alpha,6alpha-diphenylglycoluril. Biochemistry. 1978 Oct 31;17(22):4807–4817. [PubMed]
  • Molday RS, MacKenzie D. Monoclonal antibodies to rhodopsin: characterization, cross-reactivity, and application as structural probes. Biochemistry. 1983 Feb 1;22(3):653–660. [PubMed]
  • Osborn M, Weber K. Immunofluorescence and immunocytochemical procedures with affinity purified antibodies: tubulin-containing structures. Methods Cell Biol. 1982;24:97–132. [PubMed]
  • Papermaster DS, Schneider BG, Zorn MA, Kraehenbuhl JP. Immunocytochemical localization of opsin in outer segments and Golgi zones of frog photoreceptor cells. An electron microscope analysis of cross-linked albumin-embedded retinas. J Cell Biol. 1978 Apr;77(1):196–210. [PMC free article] [PubMed]
  • Papermaster DS, Schneider BG, Zorn MA, Kraehenbuhl JP. Immunocytochemical localization of a large intrinsic membrane protein to the incisures and margins of frog rod outer segment disks. J Cell Biol. 1978 Aug;78(2):415–425. [PMC free article] [PubMed]
  • Pober JS, Bitensky MW. Light-regulated enzymes of vertebrate retinal rods. Adv Cyclic Nucleotide Res. 1979;11:265–301. [PubMed]
  • Polans AS, Hermolin J, Bownds MD. Light-induced dephosphorylation of two proteins in frog rod outer segments: influence of cyclic nucleotides and calcium. J Gen Physiol. 1979 Nov;74(5):595–613. [PMC free article] [PubMed]
  • Shichi H, Somers RL. Light-dependent phosphorylation of rhodopsin. Purification and properties of rhodopsin kinase. J Biol Chem. 1978 Oct 10;253(19):7040–7046. [PubMed]
  • Soos M, Siddle K. Characterization of monoclonal antibodies directed against human thyroid stimulating hormone. J Immunol Methods. 1982;51(1):57–68. [PubMed]
  • Towbin H, Staehelin T, Gordon J. Electrophoretic transfer of proteins from polyacrylamide gels to nitrocellulose sheets: procedure and some applications. Proc Natl Acad Sci U S A. 1979 Sep;76(9):4350–4354. [PubMed]
  • Trisler GD, Schneider MD, Nirenberg M. A topographic gradient of molecules in retina can be used to identify neuron position. Proc Natl Acad Sci U S A. 1981 Apr;78(4):2145–2149. [PubMed]
  • Tzartos SJ, Rand DE, Einarson BL, Lindstrom JM. Mapping of surface structures of electrophorus acetylcholine receptor using monoclonal antibodies. J Biol Chem. 1981 Aug 25;256(16):8635–8645. [PubMed]
  • Wells JN, Kramer GL. Phosphodiesterase inhibitors as tools in cyclic nucleotide research: a precautionary comment. Mol Cell Endocrinol. 1981 Jul;23(1):1–9. [PubMed]

Articles from The Journal of General Physiology are provided here courtesy of The Rockefeller University Press