Unit

UNIT 10.8 Immunoblotting and Immunodetection

  1. Sean Gallagher1,
  2. Scott E. Winston (tank transfer systems)2,
  3. Steven A. Fuller (tank transfer systems)2,
  4. John G.R. Hurrell (tank transfer systems; reversible staining of proteins)3

Published Online: 1 JUL 2008

DOI: 10.1002/0471142727.mb1008s83

Current Protocols in Molecular Biology

Current Protocols in Molecular Biology

How to Cite

Gallagher, S., Winston (tank transfer systems), S. E., Fuller (tank transfer systems), S. A. and Hurrell (tank transfer systems; reversible staining of proteins), J. G. 2008. Immunoblotting and Immunodetection. Current Protocols in Molecular Biology. 83:III:10.8:10.8.1–10.8.28.

Author Information

  1. 1

    UVP, Inc., Upland, California

  2. 2

    Nabi, Rockville, Maryland

  3. 3

    FluorRx, Carmel, Indiana

Publication History

  1. Published Online: 1 JUL 2008
  2. Published Print: JUL 2008

Abstract

Immunoblotting (western blotting) is used to identify specific antigens recognized by polyclonal or monoclonal antibodies. This unit provides protocols for all steps, starting with solubilization of the protein samples, usually by means of SDS and reducing agents. Following solubilization, the material is separated by SDS-PAGE and the antigens are electrophoretically transferred to a membrane, a process that can be monitored by reversible staining with Ponceau S. The transferred proteins are bound to the surface of the membrane, providing access to immunodetection reagents. After nonspecific binding sites are blocked, the membrane is probed with the primary antibody and washed. The antibody-antigen complexes are tagged with horseradish peroxidase or alkaline phosphatase coupled to a secondary anti-IgG antibody, and detected using appropriate chromogenic or luminescent substrates. Finally, membranes may be stripped and reprobed. Curr. Protoc. Mol. Biol. 83:10.8.1-10.8.28. © 2008 by John Wiley & Sons, Inc.

Keywords:

  • immunoblot;
  • western blot;
  • horseradish peroxidase;
  • alkaline phosphatase;
  • antibodies