Identifying Myc Interactors

In this chapter, we discuss in detail two essential methods used to evaluate the interaction of Myc with another protein of interest: co-immunoprecipitation (Co-IP) and in vitro pull-down assays. Co-IP is a method that, by immunoaffinity, allows the identification of protein–protein interactions within cells. We provide methods to conduct Co-IPs from whole-cell extracts as well as cytoplasmic and nuclear-enriched fractions. By contrast, the pull-down assay evaluates whether a bait protein that is bound to a solid support can specifically interact with a prey protein that is in solution. We provide methods to conduct in vitro pull-downs and further detail how to use this assay to distinguish whether a protein–protein interaction is direct or indirect. We also discuss methods used to screen for Myc interactors and provide an in silico strategy to help prioritize hits for further validation using the described Co-IP and in vitro pull-down assays.