Unit

UNIT 10.10 Immunoblot Detection

  1. Sean Gallagher

Published Online: 1 MAY 2001

DOI: 10.1002/0471140864.ps1010s04

Current Protocols in Protein Science

Current Protocols in Protein Science

How to Cite

Gallagher, S. 2001. Immunoblot Detection. Current Protocols in Protein Science. 4:10.10:10.10.1–10.10.12.

Author Information

  1. Hoefer Scientific Instruments, San Francisco, California

Publication History

  1. Published Online: 1 MAY 2001
  2. Published Print: JUN 1996

Abstract

Immunoblotting (often referred to as western blotting) is used to identify specific antigens recognized by polyclonal or monoclonal antibodies. Following electrophoretic separation of proteins and transfer from the gel to an appropriate membrane, the immobilized proteins are probed with specific antibodies to identify and quantitate any antigens present. After being probed with primary antibody, the membrane is washed and the antibody-antigen complexes are identified using horseradish peroxidase (HRPO) or alkaline phosphatase (AP) enzymes coupled to the secondary anti-immunoglobulin-G (anti-IgG) antibody (e.g., goat anti-rabbit IgG). As described in this unit, the detection enzymes are attached directly or via an avidin-biotin bridge to the secondary antibody. Chromogenic or luminescent substrates are also described for visualizing the activity.