Abstract
Immunoblotting provides a simple and effective method for identifying specific antigens in a complex mixture of proteins. Initially, the constituent polypeptides are separated using sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), or a similar technique, and then are transferred either electrophoretically or by diffusion onto a nitrocellulose filter. Once immobilized on a sheet of nitrocellulose, specific polypeptides can be identified using antibodies that bind to antigens retained on the filter and subsequent visualization of the resulting antibody-antigen complex. This chapter describes conditions suitable for binding antibodies to immobilized proteins and methods for locating these antibody-antigen complexes using appropriately labeled ligands. These methods are based on those of Blake et al. (1), Burnette (2), and Towbin et al. (3).
This is a preview of subscription content, log in via an institution to check access.
Access this chapter
Tax calculation will be finalised at checkout
Purchases are for personal use only
References
Blake, M. S., Johnson, K. H., Russell-Jones, G. J., and Gotschlich, E. C. (1984) A rapid, sensitive method for detection of alkaline phosphatase-conjugated anti-antibodies on Western blots. Anal. Biochem. 136, 175–179.
Burnette, W. N. (1981) “Western blotting”: Electrophoretic transfer of proteins from sodium dodecyl sulfate-polyacrylamide gels to unmodified nitrocellulose and radiographic detection with antibody and radioiodinated protein A. Anal. Biochem. 112, 195–203.
Towbin, H., Staehelin, T., and Gordon, J. (1979) Electrophoretic transfer of proteins from polyacrylamide gels to nitrocellulose sheets: Procedure and some applications. Proc. Natl. Acad. Sci. USA 76, 4350–4354.
Tijssen, P. (1985) Practice and Theory of Enzyme Immunoassays. Elsevier, Amsterdam.
Vaessen, R. T. M. J., Kreide, J., and Groot, G. S. P. (1981) Protein transfer to nitrocellulose filters. FEBS Lett. 124, 193–196.
Towbin, H. and Gordon, J. (1984) Immunoblotting and dot immunobinding-current status and outlook. J. Immunol. Methods 72, 313–340.
Akerstrom, B., Brodin, T., Reis, K., and Bjorck, L. (1985) Protein G: A powerful tool for binding and detection of monoclonal and polyclonal antibodies. J. Immunol. 135, 2589–2592.
Langone, J. J. (1980) 125I-labeled protein A: reactivity with IgG and use as a tracer in radioimmunoassay, in Methods in Enzymology, vol. 70 (Vunakis, H. V. and Langone, J. J., eds.) Academic, New York, pp. 356–375.
Thean, E. T. and Toh, B. H. (1989) Western immunoblotting: temperature-dependent reduction in background staining. Anal. Biochem. 177, 256–258.
DenHollander, N. and Befus, D. (1989) Loss of antigens from immunoblotting membranes. J. Immunol. Methods 122, 129–135.
Hoffman, W. L., Jump, A. A., and Ruggles, A. O. (1994) Soaking nitrocellulose blots in acidic buffers improves the detection of bound antibodies without loss of biological activity. Anal. Biochem. 217, 153–155.
Miranda, P. V., Brandelli, A., and Tezon, J. G. (1993) Instantaneous blocking for immunoblots. Anal. Biochem. 209, 376,377.
Adams, J. C. (1981) Heavy metal intensification of DAB-based HRP reaction product. J. Histochem. Cytochem. 29, 775.
Esmaeli-Azad, B. and Feinstein, S. C. (1991) A general radiochemical-color method for quantitation of immunoblots. Anal. Biochem. 199, 275–278.
Author information
Authors and Affiliations
Editor information
Editors and Affiliations
Rights and permissions
Copyright information
© 1996 Humana Press Inc., Totowa, NJ
About this protocol
Cite this protocol
Kruger, N.J. (1996). Detection of Polypeptides on Blots Using Secondary Antibodies or Protein A. In: Walker, J.M. (eds) The Protein Protocols Handbook. Springer Protocols Handbooks. Humana Press. https://doi.org/10.1007/978-1-60327-259-9_48
Download citation
DOI: https://doi.org/10.1007/978-1-60327-259-9_48
Publisher Name: Humana Press
Print ISBN: 978-0-89603-338-2
Online ISBN: 978-1-60327-259-9
eBook Packages: Springer Book Archive