Laboratory of Structural and Supramolecular Chemistry, Institute of Nanoscience and Nanotechnology, National Centre for Scientific Research "DEMOKRITOS", Athens, Greece.
Centre de Biophysique Moléculaire, CNRS UPR4301, Orléans Cedex 2, France.
Methods Mol Biol. 2021;2209:73-85. doi: 10.1007/978-1-0716-0935-4_5.
Thermofluor or thermal shift assay is an easily implementable, high-throughput method for assessing the thermostability of proteins and the influence of various ligands on that stability. It is particularly useful for the assaying of ligands that may stabilize oligomeric helicases, which rely on both substrates (oligonucleotides) and nucleotide cofactors (ATP analogues) for their stability in a functional state. In this chapter, we describe the rationale and present a basic protocol for the use of this technique. Multi-ligand screening is also discussed via a worked example of the stabilization of a hexameric RNA helicase, a target protein for structural studies in our laboratories.
热荧光或热移动分析是一种易于实施、高通量的方法，可用于评估蛋白质的热稳定性和各种配体对该稳定性的影响。它对于测定可能稳定寡聚螺旋酶的配体特别有用，寡聚螺旋酶的稳定性依赖于其在功能状态下的两种底物（寡核苷酸）和核苷酸辅助因子（ATP 类似物）。在本章中，我们描述了该技术的基本原理和基本方案。通过我们实验室中结构研究的靶蛋白——六聚体 RNA 螺旋酶的稳定化这一实例，还讨论了多配体筛选。
