Purification and assay of mitotic motors.
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.
Tao
Li
L
Department of Molecular and Cell Biology, University of California, Davis, CA 95616, USA. ltao@ucdavis.edu
Scholey
Jonathan M
JM
D002850:Chromatography, Gel / Q000379:methods; D020409:Molecular Motor Proteins / Q000737:chemistry* / Q000302:isolation & purification; D008941:Spindle Apparatus
D020409:Molecular Motor Proteins