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Methods Mol Biol. 2017;1515:3-21.

A Sister Chromatid Cohesion Assay Using Xenopus Egg Extracts.

Author information

1
Chromosome Dynamics Laboratory, RIKEN, 2-1 Hirosawa, Wako, Saitama, 351-0198, Japan.
2
Chromosome Dynamics Laboratory, RIKEN, 2-1 Hirosawa, Wako, Saitama, 351-0198, Japan. hiranot@riken.jp.

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.

KEYWORDS:

Cell-free assay; Cohesin; Condensins; Immunodepletion; Immunofluorescence; Sister chromatid cohesion; Xenopus egg extracts

PMID:
27797070
DOI:
10.1007/978-1-4939-6545-8_1
[Indexed for MEDLINE]

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