Protein–Protein Interactions: Pull-Down Assays

Abstract Determining protein partners is an essential step toward understanding protein function and identifying relevant biological pathways. Many methods exist for investigating protein-protein interactions. The pull-down assay is an in vitro technique used to detect physical interactions between two or more proteins and an invaluable tool for confirming a predicted protein-protein interaction or identifying novel interacting partners. This method typically involves the use of affinity purification with various wash and elution steps. In this chapter, we describe how an interaction between two purified bacterial proteins or between bacterial and eukaryotic proteins can be detected by pull-down experiments.