Liu Yu-San, Ding Shi-You, Himmel Michael E
National Renewable Energy Laboratory, Biosciences Center, Golden, CO, USA.
Methods Mol Biol. 2012;908:129-40. doi: 10.1007/978-1-61779-956-3_13.
Single-molecule fluorescence detection is an invaluable technique for the study of molecular behavior in biological systems, both in vitro and in vivo. In this chapter, we focus on detailed protocols that utilize Total Internal Reflection Fluorescence Microscopy (TIRF-M) to visualize single molecules of carbohydrate-binding module (CBM) labeled with green fluorescent protein (GFP). The content describes step-by-step sample preparation and data acquisition, processing, and analysis. These methods can also be further used to study interactions between domains of cellulase molecules and between cellulases and cellulose.
单分子荧光检测是研究生物系统中分子行为（包括体外和体内）的一项极有价值的技术。在本章中，我们重点介绍利用全内反射荧光显微镜（TIRF-M）来可视化用绿色荧光蛋白（GFP）标记的碳水化合物结合模块（CBM）单分子的详细实验方案。内容描述了样品制备、数据采集、处理和分析的一步步过程。这些方法还可进一步用于研究纤维素酶分子各结构域之间以及纤维素酶与纤维素之间的相互作用。
