Functional analysis of the spindle-checkpoint proteins using an in vitro ubiquitination assay

The spindle checkpoint helps to ensure the fidelity of chromosome segregation during mitosis and meiosis. In response to sister chromatids not properly attached to the mitotic spindle, this checkpoint blocks the activity of a large ubiquitin protein ligase complex, called the anaphase-promoting complex (APC) or cyclosome. This chapter describes the detailed protocols of an in vitro ubiquitination assay reconstituted with purified APC, cofactors, other ubiquitination enzymes, and the spindle-checkpoint proteins. This assay is extremely useful in dissecting the biochemical functions of various spindle-checkpoint proteins.