Cite as: Cold Spring Harb. Protoc.; 2006; doi:10.1101/pdb.prot4272

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Immunoblotting: Antigen Detection Using Chromogenic Methods

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 chromogenic detection include anti-immunoglobulin antibody-coupled enzymes such as alkaline phosphatase, which, in the presence of the bromochloroindolyl phosphate/nitro blue tetrazolium (BCIP/NBT) substrate, generates an intense black-purple precipitate at the site of enzyme binding. The reaction proceeds at a steady rate, thus allowing accurate control of the development of the reaction simply by monitoring the length of incubation. Chemiluminescence (see Immunoblotting: . . . [Full Text of this Article]


MATERIALS

Reagents


METHOD


TROUBLESHOOTING


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