Co-immunoprecipitations revisited: an update on experimental concepts and their implementation for sensitive interactome investigations of endogenous proteins
2007
Markham, Kelly | Bai, Yu | Schmitt-Ulms, Gerold
The study of protein-protein interactions involving endogenous proteins frequently relies on the immunoaffinity capture of a protein of interest followed by mass spectrometry-based identification of co-purifying interactors. A notorious problem with this approach is the difficulty of distinguishing physiological interactors from unspecific binders. Additional challenges pose the need to employ a strategy that is compatible with downstream mass spectrometry and minimizes sample losses during handling steps. Finally, the complexity of data sets demands solutions for data filtering. Here we present an update on co-immunoprecipitation procedures for sensitive interactome mapping applications. We define the relevant terminology, review methodological advances that reduce sample losses, and discuss experimental strategies that facilitate recognition of candidate interactors through a combination of informative controls and data filtering. Finally, we provide starting points for initial validation experiments and propose conventions for manuscripts which report on co-immunoprecipitation work.
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