Detection of Proteins on Blots Using the Avidin-Biotin System

Abstract

Techniques of one- and two-dimensional polyacrylamide gel electrophoresis are capable of resolving complex mixtures of proteins, allowing characterization of the separated proteins in terms of their size, charge, hydrophobicity, and abundance. However, the further characterization of the separated components is dependent on the availability of other techniques. One powerful approach to this problem is to investigate the interactions of the separated proteins with specific antibodies or other ligands, such as lectins. This is best achieved by the technique of Western blotting (1) in which the separated proteins are transferred out of the gels onto the surface of a support matrix, such as nitrocellulose or polyvinylidene difluoride (PVDF). Protein transfer can be achieved by capillary, contact diffusion of vacuum techniques, but more rapid and efficient transfer is achieved if the proteins are electrophoretically removed from the gel to the support by application of an electric field perpendicular to the plane of the gel. This technique, based on the method of Towbin et al. (2), is known as electroblotting and is described in Chapter 24.