Far-Western Blotting

Protein-protein interactions play an important role in the proper functioning of the cell. Far-western blotting is a useful technique to study protein-protein interactions. In this method, proteins of interest are attached to a membrane and then probed with a non-antibody protein. While western blotting uses specific antibodies to detect target proteins, far-western blotting uses a protein probe to detect proteins based on the presence or absence of binding sites on the target protein for the probe. This method permits the study of protein-protein interactions in biological processes like signal transduction, including interactions controlled by posttranslational modification when specific modular protein-binding domains are used as probes. This chapter describes a rapid and simple protocol for far-western blotting developed by Joshua Jadwin, Bruce Mayer, and Kazuya Machida, in which they used GST-tagged Src homology 2 (SH2) domains to probe cellular proteins in a phosphorylation-dependent manner. Here, a batch quantification technique (devised and described earlier by Mayer et al.) that permits the direct comparison of probe-binding patterns is also provided.