Please cite as: CSH Protocols; 2006; doi:10.1101/pdb.prot4271

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Immunoblotting: Antigen Detection Using Chemiluminescence

Ed Harlow and David Lane

This protocol was adapted from "Immunoblotting," Chapter 8, in Using Antibodies by Ed Harlow and David Lane. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY, USA, 1999.

The first 100 words of the full text of this article appear below.


INTRODUCTION

The blot is blocked to prevent nonspecific adsorption of the immunological reagents. Antibodies are then bound to the proteins immobilized on the membrane, and the antigen is detected by labeling the antibodies with conveniently identified tags. Common labeling methods for chemiluminescent detection include anti-immunoglobulin antibody-coupled enzymes such as horseradish peroxidase, which catalyzes the oxidation of luminol and in turn releases light. This light emission can be captured on standard X-ray film to yield an excellent record that is easy to display; however, it is difficult to establish accurate quantitation of protein levels, and light emission cannot be easily controlled. For . . . [Full Text of this Article]


MATERIALS

Reagents

Equipment


METHOD


TROUBLESHOOTING


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