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

Xuelian Luo, Hongtao Yu

Research output: Contribution to journalReview article

Abstract

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.

Original languageEnglish (US)
Pages (from-to)246-255
Number of pages10
JournalMethods in Enzymology
Volume398
DOIs
StatePublished - Jan 1 2005

ASJC Scopus subject areas

  • Biochemistry
  • Molecular Biology

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