Protocol abstract
Nature Protocols 1, - 2305 - 2314 (2006)
Published online: 21 December 2006 | doi:10.1038/nprot.2006.396
Subject Categories: Biochemistry and protein analysis | Cell and developmental biology | Imaging | Isolation, purification and separation | Model organisms
Investigating mitotic spindle assembly and function in vitro using Xenopus laevis egg extracts
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 
1.5 h to prepare the extract, 1–3 h for spindle-assembly experiments and an additional 1–3 h if immunodepletion is performed.
- University of California, Berkeley, 315 Life Sciences Addition, Berkeley, CA 94720-3200, USA.
Correspondence to: Rebecca Heald1 e-mail: heald@socrates.berkeley.edu
nature-products
A-Z product listing
- 13 ml plastic tubes(Sarstedt)
- ATP(Sigma)
- Biotin-14dATP(Invitrogen)
- Biotin-21dUTP(Clontech)
- bovine serum albumin(Sigma)
- creatine phosphate(Roche Diagnostics)
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