Figure 3.

Analysis of Mad2 complexes during an active checkpoint. HeLa cells arrested with nocodazole were harvested by mitotic shake-off and a cell extract was prepared. The total extract was loaded onto a Superdex 200 column following clarification by centrifugation and 500 μl fractions collected and analyzed by SDS-PAGE and western blot (Input). Mad2 complexes were isolated from each fraction by immunopurification with the pan-Mad2 antibody and analyzed by protein gel blot (αpan-Mad2). A similar purification was done with the C-Mad2 specific antibody (αC-Mad2) or O-Mad2 specific antibody (αO-Mad2).