Detection of Polypeptides on Immunoblots Using Secondary Antibodies or 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. Once immobilized on a sheet of nitrocellulose, specific polypeptides can be identified using antibodies that bind to antigens retained on the filter and subsequent visualization of the resulting antibody-antigen complex. This chapter describes conditions suitable for binding antibodies to immobilized proteins and methods for locating these antibody-antigen complexes using appropriately labeled ligands. These methods are based on those of Blake et al. (1), Burnette (2), and Towbin et al. (3).