Hänselmann Siegfried, Herten Dirk-Peter
Physikalisch-Chemisches Institut & CellNetworks Cluster, Heidelberg University, Im Neuenheimer Feld 229, 69120, Heidelberg, Germany.
Methods Mol Biol. 2017;1663:127-138. doi: 10.1007/978-1-4939-7265-4_11.
Measuring the kinetics of protein-protein interactions between molecules in the plasma membrane of live cells provides valuable information for understanding dynamic processes, like cellular signaling, on a molecular scale. Two-color single-molecule tracking is a fluorescence microscopy-based method to detect and quantify specific protein-protein interactions on a single-event level, providing sensitivity to heterogeneities and rare events. Fundamentally, it allows following the movement of single molecules of two different protein species in live cells with a localization precision beyond the diffraction limit of light in real time. It hence provides information about the diffusion behavior of every protein as well as about their dimerization kinetics. Here, we describe all the necessary steps to obtain two-color tracking data of plasma membrane-associated proteins in live cells using SNAP-tag and HaloTag fusion constructs and total internal reflection fluorescence (TIRF) microscopy. Also, we outline the main steps needed for analyzing the recorded data.
测量活细胞质膜中分子间蛋白质 - 蛋白质相互作用的动力学，可为在分子尺度上理解细胞信号传导等动态过程提供有价值的信息。双色单分子追踪是一种基于荧光显微镜的方法，用于在单事件水平上检测和量化特定的蛋白质 - 蛋白质相互作用，对异质性和罕见事件具有敏感性。从根本上讲，它能够实时追踪活细胞中两种不同蛋白质种类的单分子运动，其定位精度超出了光的衍射极限。因此，它提供了关于每种蛋白质扩散行为及其二聚化动力学的信息。在这里，我们描述了使用SNAP标签和Halo标签融合构建体以及全内反射荧光（TIRF）显微镜在活细胞中获取质膜相关蛋白质双色追踪数据的所有必要步骤。此外，我们概述了分析记录数据所需的主要步骤。
