Western Blot Analysis

Neuroscience has quickly evolved to include classical neurochemical as well as molecular approaches to the study of proteins, This marriage of disciplines has been brought about not only by our need to further explore systems of specific interest, but also by the increased availability of commercial products and equipment. Protocols for the study of proteins are also numerous, and their use varies according to the experimental objectives. One of these approaches to the study of proteins, Western blot analysis —also known as electroblotting (Towbin et al., 1979) or immunoblotting (Burnette, 1981)—is a rapid and sensitive assay which combines the resolution of gel electrophoresis and the principles of immunological recognition of an antigen (e.g., the protein) by an appropriate antibody. We will begin with a discussion of the general principles and considerations of gel electrophoresis, the preparation of cell extracts, and the choice of antibodies, followed by a detailed description of the most common and reliable procedures that we have adapted and use routinely and satisfactorily. The reader wishing greater detail about alternate protocols is directed to other available sources (Harlow and Lane, 1988; Timmons and Dunbar, 1990; Ausubel et al., 1994).