Methods Mol Biol 2009 Jan 01;545:221-32. doi: 10.1007/978-1-60327-993-2_13.

Probing kinetochore structure and function using Xenopus laevis frog egg extracts.

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Kinetochores are multiprotein machines that initiate mitotic checkpoint signaling and control chromosome movement through interactions with microtubules. Our lab has utilized Xenopus laevis frog egg extracts to investigate the requirements for kinetochore assembly and disassembly in vertebrates. Egg extracts support the assembly of functional kinetochores that are capable of binding microtubules, aligning and segregating chromosomes, and sending spindle checkpoint signals. This is the only in vitro system that assembles functional kinetochores, making it particularly well suited for these types of studies. Probing kinetochore assembly using the biochemically tractable egg extract system has elucidated the intricate assembly requirements for numerous vertebrate kinetochore proteins. The following techniques have been used to characterize kinetochore assembly requirements. In addition, we describe assays that we utilized to identify factors that promote maintenance of preassembled kinetochores and those that induce kinetochore disassembly.

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