Purification and assay of mitotic motors

Li Tao, Jonathan M. Scholey

Research output: Contribution to journalArticle

3 Scopus citations

Abstract

To understand how mitotic kinesins contribute to the assembly and function of the mitotic spindle, we need to purify these motors and analyze their biochemical and ultrastructural properties. Here we briefly review our use of microtubule (MT) affinity and biochemical fractionation to obtain information about the oligomeric state of native mitotic kinesin holoenzymes from eggs and early embryos. We then detail the methods we use to purify full length recombinant Drosophila embryo mitotic kinesins, using the baculovirus expression system, in sufficient yields for detailed in vitro assays. These two approaches provide complementary biochemical information on the basic properties of these key mitotic proteins, and permit assays of critical motor activities, such as MT-MT crosslinking and sliding, that are not revealed by assaying motor domain subfragments.

Original languageEnglish (US)
Pages (from-to)233-241
Number of pages9
JournalMethods
Volume51
Issue number2
DOIs
StatePublished - Jun 2010

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Keywords

  • Baculovirus
  • In vitro motility assays
  • Mitotic motor
  • Protein purification

ASJC Scopus subject areas

  • Molecular Biology
  • Biochemistry, Genetics and Molecular Biology(all)

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