Protein–Protein Interactions: Co-immunoprecipitation

Proteins often do not function as single substances but rather as team players in a dynamic network. Growing evidences show that protein–protein interactions are crucial in many biological processes in living cells. Genetic (such as yeast two hybrid, Y2H) and biochemical (such as co-immunoprecipitation, co-IP) methods are the commonly used methods to identify the interacting proteins. Immunoprecipitation (IP), a method using a target protein-specific antibody in conjunction with Protein A/G affinity beads, is a powerful tool to identify the molecules interacting with specific proteins. Therefore, co-IP is considered to be one of the standard methods to identify and/or confirm the occurrence of the protein–protein interaction events in vivo. The co-IP experiments can identify proteins via direct or indirect interactions or in a protein complex. Here, we use two different co-Ip protocols as an example to describe the principle, procedure, and experimental problems of co-IP. First, we show the interaction of two Agrobacterium type VI secretion system (T6SS) sheath components TssB and TssC41, and secondly, we show the protocol we used for determining the interaction of an epitope-tagged T6SS effector, Tde1 expressed in Agrobacterium with endogenously expressing adaptor/chaperone protein Tap1.