有丝分裂马达的纯化和检测。
Purification and assay of mitotic motors.
机构信息
Department of Molecular and Cell Biology, University of California, Davis, CA 95616, USA.
出版信息
Methods. 2010 Jun;51(2):233-41. doi: 10.1016/j.ymeth.2010.01.019. Epub 2010 Jan 22.
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.
为了了解有丝分裂驱动蛋白如何有助于有丝分裂纺锤体的组装和功能，我们需要纯化这些马达并分析它们的生化和超微结构特性。在这里，我们简要回顾了我们使用微管（MT）亲和性和生化分级分离来获取有关卵和早期胚胎中原位有丝分裂驱动蛋白全酶的寡聚状态的信息。然后，我们详细介绍了我们使用杆状病毒表达系统在足够的产量下纯化全长重组果蝇胚胎有丝分裂驱动蛋白的方法，用于详细的体外测定。这两种方法都提供了有关这些关键有丝分裂蛋白基本特性的互补生化信息，并允许测定关键的马达活性，例如 MT-MT 交联和滑动，而通过测定马达结构域亚片段则无法揭示这些活性。
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