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.
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Hannak, E., Heald, R. Investigating mitotic spindle assembly and function in vitro using Xenopus laevis egg extracts. Nat Protoc 1, 2305–2314 (2006). https://doi.org/10.1038/nprot.2006.396
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DOI: https://doi.org/10.1038/nprot.2006.396
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