Proteins often do not function as single substances but rather as team players in a dynamic network. Growing evidence shows 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 methods commonly used at the beginning of a study 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 molecules that interact with specific proteins. Therefore, co-IP is considered to be one of the standard methods of identifying or confirming the occurrence of protein–protein interaction events in vivo. Co-IP experiments can identify proteins via direct or indirect interactions or in a protein complex. Here, we use Agrobacterium type VI secretion system (T6SS) sheath components TssB-TssC41 interaction as an example to describe the principle, procedure, and experimental problems of co-IP.
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This work was supported by a research grant from the Taiwan Ministry of Science and Technology (MOST 104-2311-B-001-025 -MY3) to E.M. Lai. J.S. Lin is the recipient of postdoctoral fellowships from Academia Sinica.
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