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Nat Protoc. 2006;1(5):2305-14.

Investigating mitotic spindle assembly and function in vitro using Xenopus laevis egg extracts.

Author information

1
University of California, Berkeley, 315 Life Sciences Addition, Berkeley, CA 94720-3200, USA.

Abstract

Extracts from Xenopus laevis eggs provide a powerful system for the study of cell division processes in vitro through biochemical reconstitution and manipulation, and microscopic analysis. We provide protocols for the preparation of metaphase-arrested extracts and in vitro assays to examine the following pathways of spindle assembly: 1) Sperm nuclei added to meiotic extracts, supporting the formation of half-spindles and bipolar spindle structures around unreplicated chromosomes; 2) sperm nuclei added to extracts that cycle through interphase and form spindles that are capable of undergoing anaphase and chromosome segregation; and 3) spindle formation around chromatin-coated beads. Finally, we describe methods to inhibit a specific protein by immunodepletion or addition of an inhibitor such as a dominant-negative construct. These techniques can be used to analyze the mitotic function of a given protein. It takes approximately 1.5 h to prepare the extract, 1-3 h for spindle-assembly experiments and an additional 1-3 h if immunodepletion is performed.

PMID:
17406472
DOI:
10.1038/nprot.2006.396
[Indexed for MEDLINE]

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