UNIT 8.10 Immunoblotting and Immunodetection
Published Online: 1 MAY 2001
Copyright © 2003 by John Wiley and Sons, Inc.
Lab Protocol Title
Current Protocols in Immunology
How to Cite
Gallagher, S., Winston, S. E., Fuller, S. A. and Hurrell, J. G. 2001. Immunoblotting and Immunodetection. Current Protocols in Immunology. 26:IV:8.10:8.10.1–8.10.21.
- Published Online: 1 MAY 2001
- Published Print: JUN 1998
This is not the most recent version of the article. View current version (1 NOV 2008)
Immunoblotting (often referred to as western blotting) is used to identify specific antigens recognized by polyclonal or monoclonal antibodies. This unit presents procedures for electrophoretically transferring antigens from a denaturing polyacrylamide gel in a tank or a semidry transfer apparatus to a nitrocellulose, PVDF, or nylon membrane. The process can be monitored by reversible staining or by Ponceau S staining, both of which are described here. A protocol for blotting previously stained gels is also described. The transferred proteins are bound to the surface of the membrane, providing access for reaction with immunodetection reagents. All remaining binding sites are blocked by immersing the membrane in a solution containing either a protein or detergent blocking agent. After probing with the primary antibody, the membrane is washed and the antibody-antigen complexes are identified with horseradish peroxidase (HRPO) or alkaline phosphatase enzymes coupled to the secondary anti-IgG antibody (e.g., goat anti-rabbit IgG). The enzymes are attached directly or via an avidin-biotin bridge to the secondary antibody, and protocols are provided for both methods. Chromogenic or luminescent substrates are then used as described to visualize the activity. Finally, a method for stripping and reprobing membranes is presented.