Spudich James A, Rice Sarah E, Rock Ronald S, Purcell Thomas J, Warrick Hans M
Cold Spring Harb Protoc. 2011 Nov 1;2011(11):1372-4. doi: 10.1101/pdb.prot066688.
In vitro motility assays enabled the analysis of coupling between ATP hydrolysis and movement of myosin along actin filaments or kinesin along microtubules. Single-molecule assays using laser trapping have been used to obtain more detailed information about kinesins, myosins, and processive DNA enzymes. The combination of in vitro motility assays with laser-trap measurements has revealed detailed dynamic structural changes associated with the ATPase cycle. This protocol describes the preparation of biotin-actin filaments and coverslips coated with polystyrene beads. These are then used in optical trapping dumbbell assays to study interactions between motors and filaments.
体外运动分析能够分析ATP水解与肌球蛋白沿肌动蛋白丝运动或驱动蛋白沿微管运动之间的偶联。使用激光捕获的单分子分析已用于获取有关驱动蛋白、肌球蛋白和进行性DNA酶的更详细信息。体外运动分析与激光捕获测量的结合揭示了与ATP酶循环相关的详细动态结构变化。本方案描述了生物素化肌动蛋白丝和涂有聚苯乙烯珠的盖玻片的制备。然后将它们用于光镊哑铃分析，以研究马达与细丝之间的相互作用。
