Advertisement

Nonradioactive Photoaffinity Labeling of Steroid Receptors Using Western Blot Detection System

  • Simon J. Evans
  • Frank L. Moore
Part of the Methods in Molecular Biology™ book series (MIMB, volume 176)

Abstract

Photoaffinity labeling is the process of covalently crosslinking a photoactive ligand, which can be detected postcoupling to a receptor or binding protein. This technique has proven useful in the identification of novel proteins or to further study and characterize known proteins. Photoaffinity labeling of novel receptors and binding proteins can provide information about biochemical properties of the target protein by allowing it to be traced through various separation schemes. For example, visualizing a labeled protein following one- or two-dimensional sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) can provide information about its apparent molecular weight (mol wt) and isoelectric point (1, 2). Knowledge of these biochemical characteristics, and others, can be useful in further design of purification strategies. Photoaffinity labeling of known receptors or binding proteins can be used to study important residues in the target protein’s binding site. For example, photoaffinity labeling, followed by an enzymatic digest and sequencing of the labeled peptide(s), allows the identification of specific labeled residues in or near the binding pocket (3) Other studies might also use photoaffinity labels to induce a chronic activation or inactivation of a receptor by crosslinking the ligand to the binding site.

Keywords

Chemiluminescent Detection System Radioligand Binding Assay Photoaffinity Label Nonspecific Label Western Blot Procedure 
These keywords were added by machine and not by the authors. This process is experimental and the keywords may be updated as the learning algorithm improves.

References

  1. 1.
    Wehling, M., Eisen, C., Aktas, J., Christ, M., and Theisen, K. (1992) Photoaffinity labeling of plasma membrane receptors for aldosterone from human mononuclear leukocytes. Biochem. Biophys. Res. Commun. 189, 1424–1428.PubMedCrossRefGoogle Scholar
  2. 2.
    Evans, S. J., Murray, T. F., and Moore, F. L. (2000) Partial purification and biochemical characterization of a membrane glucocorticoid receptor from an amphibian brain. J. Steroid Biochem. Mol. Biol. 72, 209–221.PubMedCrossRefGoogle Scholar
  3. 3.
    Grenot, C., Blachere, T., Rolland de Ravel, M., Mappus, E., and Cuilleron, C. Y. (1994) Identification of Trp-371 as the main site of specific photoaffinity labeling of corticosteroid binding globulin using ä6 derivatives of cortisol, corticosterone, and progesterone as unsubstituted photoreagents. Biochemistry 33, 8969–8981.PubMedCrossRefGoogle Scholar
  4. 4.
    Katzenellenbogen, J. A. and Katzenellenbogen, B. S. (1984) Affinity labeling of receptors for steroid and thyroid hormones. Vitam. Horm. 41, 213–274.PubMedCrossRefGoogle Scholar
  5. 5.
    Timmons, T. M. and Dunbar, S. D. (1990) Protein blotting and immunodetection, in Guide To Protein Purification, vol. 182. Methods in Enzymology (Deutscher M. P. ed.), Academic Press, San Diego, CA, pp. 679–688.CrossRefGoogle Scholar
  6. 6.
    Fido, R. J., Tatham, A. S., and Shewry, P. R. (1995) Western blotting analysis. Methods Mol. Biol. 49, 423–437.PubMedGoogle Scholar
  7. 7.
    Egger, D. and Bienz, K. (1994) Protein (western) blotting. Mol. Biotechnol. 1, 289–305.PubMedCrossRefGoogle Scholar
  8. 8.
    Hicks, G. R., Rayle, D. L., Jones, A. M., and Lomax, T. L. (1989) Specific photaffinity labeling of two plasma membrane polypeptides with an azido auxin. Proc. Natl. Acad. Sci. USA 86, 4948–4952.PubMedCrossRefGoogle Scholar

Copyright information

© Humana Press Inc. 2001

Authors and Affiliations

  • Simon J. Evans
    • 1
  • Frank L. Moore
    • 2
  1. 1.Mental Health Research InstituteUniversity of MichiganAnn Arbor
  2. 2.Department of ZoologyOregon State UniversityCorvallis

Personalised recommendations