Topf Andrea, Franz Peter, Tsiavaliaris Georgios
Institute for Biophysical Chemistry, Hannover Medical School, Hannover, Germany.
Biotechniques. 2017 Oct 1;63(4):187-190. doi: 10.2144/000114599.
Here, we present a MicroScale Thermophoresis (MST)-based assay for in vitro assessment of actin polymerization. By monitoring the thermophoretic behavior of ATTO488-labeled actin in a temperature gradient over time, we could follow polymerization in real time and resolve its three characteristic phases: nucleation, elongation, and steady-state equilibration. Titration experiments allowed us to evaluate the effects of actin-binding proteins (ABPs) on polymerization, including DNase I-induced inhibition and mDia2FH1FH2 (mDia2)-assisted acceleration of nucleation. The corresponding rates of actin filament elongation were quantitatively determined, yielding values in good agreement with those obtained using the pyrene-actin polymerization assay. Finally, we measured the effect of myosin on actin polymerization, circumventing the problems of fluorescence quenching and signal disturbance that occur with other techniques. MST is a simple and valuable research tool for investigating actin kinetics covering a wide range of molecular interactions, with low protein consumption.
在此，我们展示了一种基于微量热泳（MST）的方法，用于体外评估肌动蛋白聚合。通过监测ATTO488标记的肌动蛋白在温度梯度下随时间的热泳行为，我们能够实时跟踪聚合过程，并解析其三个特征阶段：成核、伸长和稳态平衡。滴定实验使我们能够评估肌动蛋白结合蛋白（ABP）对聚合的影响，包括DNase I诱导的抑制作用以及mDia2FH1FH2（mDia2）辅助的成核加速作用。定量测定了肌动蛋白丝伸长的相应速率，所得值与使用芘-肌动蛋白聚合测定法获得的值高度一致。最后，我们测量了肌球蛋白对肌动蛋白聚合的影响，避免了其他技术中出现的荧光猝灭和信号干扰问题。MST是一种简单且有价值的研究工具，用于研究涵盖广泛分子相互作用的肌动蛋白动力学，且蛋白质消耗低。
