Methods Enzymol 2005 Jan 01;398:246-55. doi: 10.1016/S0076-6879(05)98020-8.

Purification and assay of Mad2: a two-state inhibitor of anaphase-promoting complex/cyclosome.

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To maintain the fidelity of chromosome inheritance, cells utilize a surveillance mechanism called the spindle checkpoint to sense improper attachment of sister chromatids to the mitotic spindle prior to chromosome segregation. The target of the spindle checkpoint is a ubiquitin ligase called the anaphase-promoting complex or cyclosome (APC/C). The spindle checkpoint protein Mad2 inhibits the activity of APC/C through direct binding to its activator Cdc20. Studies have shown that Mad2 has two distinct natively folded conformations and that the unusual two-state behavior of Mad2 plays a crucial role in checkpoint signaling. This article describes methods for the purification of the two Mad2 conformers and for the analysis of their activities in APC/C inhibition in Xenopus egg extracts.

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Species referenced: Xenopus laevis
Genes referenced: cdc20 mad2l1