Murad Fabronia, Garcia-Saez Ana J
Interfaculty Institute of Biochemistry, University of Tübingen, Tübingen, Germany.
Methods Mol Biol. 2019;1877:337-350. doi: 10.1007/978-1-4939-8861-7_20.
The proteins of the Bcl-2 family regulate apoptosis by forming a complex interaction network whose output determines whether mitochondrial outer membrane permeabilization is executed. Quantification of complex formation between Bcl-2 proteins in solution and in membranes is therefore key to understand how the hierarchy of interactions controls cell death induction. Fluorescence correlation spectroscopy (FCS) is a noninvasive, nondestructive method to investigate the mobility and the association of fluorescently labeled biomolecules that has provided useful insight into the binding affinity of the Bcl-2 interactome. FCS is based on the detection of fluorescence fluctuations caused by the diffusion of individual molecules through a very tiny observation volume of the detection system. Scanning FCS (SFCS) solves some of the practical challenges of acquiring FCS in membranes and expands the application scope of the method. In this chapter, we explain the principle of FCS and describe protocols how it can be used to quantify interactions between Bcl-2 proteins in solution and in model membrane systems.
Bcl-2家族蛋白通过形成一个复杂的相互作用网络来调节细胞凋亡，该网络的输出决定了线粒体外膜通透性是否会发生。因此，量化溶液和膜中Bcl-2蛋白之间的复合物形成对于理解相互作用的层级如何控制细胞死亡诱导至关重要。荧光相关光谱法（FCS）是一种用于研究荧光标记生物分子的流动性和缔合的非侵入性、非破坏性方法，它为Bcl-2相互作用组的结合亲和力提供了有用的见解。FCS基于检测单个分子通过检测系统的非常微小的观察体积扩散所引起的荧光波动。扫描FCS（SFCS）解决了在膜中获取FCS的一些实际挑战，并扩展了该方法的应用范围。在本章中，我们解释了FCS的原理，并描述了如何使用它来量化溶液和模型膜系统中Bcl-2蛋白之间相互作用的方案。
