A sister chromatid cohesion assay using Xenopus egg extracts

Abstract

Cell-free extracts made from Xenopus laevis eggs enable us to recapitulate many chromosomal events associated with cell cycle progression in a test tube. When sperm chromatin is incubated with these extracts, it is first duplicated within an assembled nucleus, and is then transformed into mitotic chromosomes, in each of which sister chromatids are juxtaposed with each other in a cohesin-dependent manner. Here we describe our protocols for assembling duplicated chromosomes using egg extracts, along with cytological and biochemical assays for addressing the molecular mechanisms of sister chromatid cohesion. A powerful approach involving immunodepletion of cohesin and its regulators is also included.