Meyer D, Rines D R, Kashina A, Cole D G, Scholey J M
Section of Molecular and Cellular Biology, University of California, Davis 95616, USA.
Methods Enzymol. 1998;298:133-54. doi: 10.1016/s0076-6879(98)98015-6.
Several kinesin holoenzymes, including the heterotrimeric kinesin-II and bipolar KLP61F complexes described here, are being purified in our laboratory using microtubule affinity precipitation and conventional biochemical fractionation procedures. These protocols have been optimized by using pan-kinesin peptide antibodies and subunit-specific antibodies to monitor the enrichment of kinesin-related polypeptides in particular fractions by immunoblotting. Protein purification represents the most direct route available for determining the oligomeric state and subunit composition of a kinesin holoenzyme, for identifying tightly associated accessory subunits such as SpKAP115, and for determining the molecular architecture and functional properties of native kinesin motors. Protein purification methods therefore represent an important complementary approach to molecular genetic approaches that are being pursued in many other laboratories.
包括本文所述的异源三聚体驱动蛋白-II和双极KLP61F复合物在内的几种驱动蛋白全酶，正在我们实验室通过微管亲和沉淀和传统生化分级分离程序进行纯化。这些方案通过使用泛驱动蛋白肽抗体和亚基特异性抗体进行了优化，以通过免疫印迹监测特定级分中驱动蛋白相关多肽的富集情况。蛋白质纯化是确定驱动蛋白全酶的寡聚状态和亚基组成、鉴定紧密相关的辅助亚基（如SpKAP115）以及确定天然驱动蛋白马达的分子结构和功能特性的最直接途径。因此，蛋白质纯化方法是许多其他实验室正在采用的分子遗传学方法的重要补充方法。
