Abstract
Western blotting is employed for the detection of proteins and other macromolecules immobilized on nitrocellulose membranes. This rapid and sensitive method enables the identification and quantification of a specific protein from cell lysates or a mixture of proteins. Following the resolution on denaturing sodium dodecyl sulfate (SDS)-polyacrylamide gel electrophoresis (SDS-PAGE), the constituent polypeptides are transferred electrophoretically to a nitrocellulose membrane (1). The membrane is incubated in a solution containing primary antibody and the resultant antigen-antibody complex is detected by using appropriately labeled ligands. The most common method is based on the enzyme-linked immunodetection of antigen-specific antibodies using anti-IgG secondary antibodies conjugated with either horseradish peroxidase (HRP) or alkaline phosphatase (AP). Visualization of antibodyantigen complex is achieved through the use of an enhanced chemiluminiscent (ECL) method. It is possible to detect as little as 10-50 ng of protein with AP-conjugated secondary antibody and 0.5-1.0 ng of protein with HRP-conjugated secondary antibody using high affinity and high titer primary antibodies.
This is a preview of subscription content, log in via an institution.
Buying options
Tax calculation will be finalised at checkout
Purchases are for personal use only
Learn about institutional subscriptionsSpringer Nature is developing a new tool to find and evaluate Protocols. Learn more
References
Towbin, H., Staehelin, T., and Gordon, J. (1979) Electrophoretic transfer of proteins from polyacrylamide gels to nitrocellulose sheets: procedure and some application. Proc. Natl. Acad. Sci. USA 76, 4350–4354.
Bradford, M. M. (1976) A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. Anal. Biochem. 72, 248–254.
Laemmli, U. K. (1970) Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature 227, 680–685.
Whitehead, T. P., Kricka, L. J., Carter, T. J., and Thorpe, G. H. (1979) Analytical luminescence: its potential in the clinical laboratory. Clin. Chem. 25, 1531–1546.
Paxton, W. G., Runge, M., Horaist, C., Cohen, C., Alexander, R. W., and Bernstein, K. E. (1993) Immunohistochemical localization of rat angiotensin II AT1 receptor. Am. J. Physiol. 264, F989–F995.
Marrero, M. B., Schieffer, B., Li, B., Sun, J., Harp, J. B., and Ling, B. N. (1997) Role of Janus kinase/signal transducer and activator of transcription and mitogenactivated protein kinase cascades in angiotensin II-and platelet-derived growth factor-induced vascular smooth muscle cell proliferation. J. Biol. Chem. 272, 24,684–24,690.
Schagger, H. and von Jagow, G. (1987) Tricine-sodium dodecyl sulfate-polyacrylamide gel electrophoresis for the separation of proteins in the range from 1 to 100 kDa. Anal. Biochem. 166, 368–379.
Peluso, R. W. and Rosenberg, G. H. (1987) Quantitative electrotransfer of proteins from sodium dodecyl sulfate-polyacrylamide gels onto positively charged nylon membranes. Anal. Biochem. 162, 389–398.
Thean, E. T. and Toh, B. H. (1989) Western Blotting: temperature dependent reduction in background staining. Anal. Biochem. 177, 256–258.
Kruger, N. J. (1996) Detection of polypeptides on blots using secondary antibodies or protein A, in The Protein Protocols Handbook (Walker, J. M., ed.) Humana, Totowa, NJ, pp. 313–321.
Author information
Authors and Affiliations
Editor information
Editors and Affiliations
Rights and permissions
Copyright information
© 2001 Humana Press Inc.
About this protocol
Cite this protocol
Madamanchi, N.R., Runge, M.S. (2001). Western Blotting. In: Wang, D.H. (eds) Angiotensin Protocols. Methods in Molecular Medicine™, vol 51. Humana Press. https://doi.org/10.1385/1-59259-087-X:245
Download citation
DOI: https://doi.org/10.1385/1-59259-087-X:245
Publisher Name: Humana Press
Print ISBN: 978-0-89603-763-2
Online ISBN: 978-1-59259-087-2
eBook Packages: Springer Protocols