Department of Chemical Science and Technologies, University of Rome Tor Vergata, Rome, Italy.
Methods Mol Biol. 2023;2705:93-112. doi: 10.1007/978-1-0716-3393-9_6.
Fluorescence anisotropy (or polarization) is a powerful technique to study biomolecular association processes, by following the rotational motions of one of the two partners in the interaction, labeled with a fluorophore. It can be used to determine dissociation constants in solution, down to nM values, and unlabeled ligands can be characterized, too, by using competition experiments. In this chapter, we introduce the basic principles of the technique, compare it with other experimental approaches, and discuss the experimental details with specific examples regarding SH2 domain/phosphopeptide association processes. The experimental protocols to be used in binding experiments and displacement studies are described, as well as the caveats to be considered in performing accurate measurements.
荧光各向异性（或偏振）是一种强大的技术，可通过跟踪相互作用中标记荧光团的两个伴侣之一的旋转运动，来研究生物分子的结合过程。它可用于确定溶液中的离解常数，低至纳摩尔值，并且可以通过使用竞争实验来表征未标记的配体。在本章中，我们介绍了该技术的基本原理，将其与其他实验方法进行了比较，并结合有关 SH2 结构域/磷酸肽结合过程的具体实例讨论了实验细节。描述了用于结合实验和置换研究的实验方案，以及在进行准确测量时需要考虑的注意事项。
