Localization and Characterization of Protein-Protein Interaction Sites

This chapter aims to describe methods to identify and characterize protein-protein interactions that were developed during our studies on translation initiation factor complexes. Methods include the two-hybrid assay, the GST pull-down assay, and the coimmunoprecipitation (co-IP) assay. The two-hybrid assay provides for a convenient start to find the minimal interaction domains, which generally produce well-behaved recombinant proteins suited for various in vitro interaction assays. Emphasis is placed on demonstrating physiological relevance of identified interactions. The effective strategy is to find mutations that reduce the interaction by genetic or site-directed mutational approaches and obtain correlations between their effects in vitro (GST pull down) and effects in vivo (co-IP).