Identification of protein interactions by far western analysis.

This unit describes far western blotting, a method of identifying protein-protein interactions. In a far western blot, one protein of interest is immobilized on a solid support membrane, then probed with a non-antibody protein. As described, far western blots can be used to identify specific interacting proteins in a complex mixture of proteins. They are particularly useful for examining interactions between proteins that are difficult to analyze by other methods due to solubility problems or because they are difficult to express in cells. This method is performed totally in vitro, and the proteins of interest can be prepared in a variety of ways. A protocol is also provided for determining the effects of specific peptide residues or post-translational modifications on protein-protein interactions. Many different detection techniques, either radioactive or nonradioactive, can be used. For example, the protein probe may be detected indirectly with an antibody, rather than being labeled radioactively.