Spudich James A, Rice Sarah E, Rock Ronald S, Purcell Thomas J, Warrick Hans M
Cold Spring Harb Protoc. 2011 Nov 1;2011(11):1370-1. doi: 10.1101/pdb.prot066670.
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 a method for attaching anti-GFP (green fluorescent protein) antibodies to microspheres. GFP-motor fusion proteins can then be adsorbed to the microspheres for use in single-molecule motility studies and optical trapping experiments.
体外运动分析能够分析ATP水解与肌球蛋白沿肌动蛋白丝的运动或驱动蛋白沿微管的运动之间的偶联。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。本方案描述了一种将抗绿色荧光蛋白(GFP)抗体连接到微球上的方法。然后可以将GFP-马达融合蛋白吸附到微球上,用于单分子运动研究和光镊实验。