Immunodetection of Proteins on “Western” Blots Using ¹²⁵I-Labeled Protein A

Abstract

Immunoblotting provides a simple and effective method for identifying specific antigens in a complex mixture of proteins. Initially, the constituent polypeptides are separated using SDS-polyacrylamide gel electrophoresis, or a similar technique, and then are transferred either electrophoretically or by diffusion onto a nitrocellulose filter. The transfer of proteins from gels onto membranes is discussed in vol. 1 of this series and Chapters 28 and 29, this volume. Once immobilized on a sheet of nitrocellulose, specific polypeptides can be identified using antibodies that bind to antigens retained on the filter. In this chapter we describe conditions appropriate for binding antibodies to immobilized proteins and a method for locating these antibody-antigen complexes using ¹²⁵I-labeled protein A. These methods are based on those of Burnett (1) and Towbin et al. (2).