Determination of Protein-Protein Interactions and the Motifs That Mediate Them

Abstract

Determining whether or not a specific protein interacts with another can be accomplished in a number of ways, which can be divided into in vivo or in vitro approaches. The in vivo approaches include yeast two-hybrid, yeast threehybrid, mammalian two-hybrid, one-hybrid, and FRET analyses. While the most common in vitro approaches are glutathione-S-transferase (GST) pulldown assays, co-immunoprecipitation, immune depletion, gel-filtration or sucrose (or glycerol)-density gradient analysis, far-Western blot analysis, and chemical crosslinking. Each one of these assays has strengths and weaknesses and usually a combination of methods can lead a compelling case that a given interaction actually can occur within the cell. Many of these approaches utilize what can be considered transient overexpression of the proteins being studied and this can lead to interactions occurring that do not normally occur under physiologic conditions. Also, upon overexpression a protein may be found in subcellular compartments where they do not normally exist. For example, upon overexpression, a normally cytosolic protein may be found in significant concentrations in the nucleus. Nevertheless, the use of cellular overexpression systems usually yields information that is physiologically relevant. The following section outlines the various approaches that can be taken to document that one protein is capable of interacting with another.