Purification of the Mitotic Checkpoint Complex, an Inhibitor of the APC/C From HeLa Cells

The mitotic checkpoint prevents cells with unaligned chromosomes from prematurely exiting mitosis by inhibiting the anaphase-promoting complex/cyclosome (APC/C) from targeting key proteins for ubiquitin-mediated proteolysis. Great efforts have been made recently to identify and characterize the factors involved in this checkpoint pathway. These studies will ultimately lead to a better understanding of cell cycle defects and chromosome instability. We report here a protocol for purification of an inhibitor of the APC/C from HeLa cells, called mitotic checkpoint complex (MCC). Our procedure is based on biochemical purification and characterization of the APC/C inhibitory activity from extracts of HeLa cells.