Ehrhardt Maximilian K G, Warring Suzanne L, Gerth Monica L
Department of Biochemistry, University of Otago, Dunedin, New Zealand.
Department of Biological Sciences, Victoria University of Wellington, Wellington, New Zealand.
Methods Mol Biol. 2018;1729:281-290. doi: 10.1007/978-1-4939-7577-8_22.
Identifying the ligands sensed by chemoreceptors remains challenging, in part because current screening methods are low-throughput, costly, and/or time-consuming. In contrast, fluorescence thermal shift (FTS) assays provide a fast and inexpensive approach to chemoreceptor-ligand screening. In FTS assays, the temperature at which a protein denatures is measured by monitoring the fluorescence of a dye with affinity for hydrophobic regions of the protein, which are exposed as the protein unfolds. A detectable increase (or "shift") in the melting temperature (T ) of the protein in the presence of a potential ligand indicates binding. Here, we present our protocol for using FTS assays for the screening of chemoreceptor ligands in a high-throughput, 96-well plate format. We have also included details on the use of Biolog Phenotype Microarray plates as a convenient ligand library, although the methods described should be generally applicable to other library formats as well.
识别化学感受器所感知的配体仍然具有挑战性，部分原因是当前的筛选方法通量低、成本高且/或耗时。相比之下，荧光热位移（FTS）分析为化学感受器-配体筛选提供了一种快速且廉价的方法。在FTS分析中，通过监测与蛋白质疏水区域具有亲和力的染料的荧光来测量蛋白质变性的温度，随着蛋白质展开，这些疏水区域会暴露出来。在存在潜在配体的情况下，蛋白质解链温度（Tm）出现可检测到的升高（或“位移”）表明发生了结合。在此，我们展示了使用FTS分析以高通量96孔板形式筛选化学感受器配体的方案。我们还详细介绍了使用Biolog表型微阵列板作为便捷的配体库，尽管所描述的方法通常也适用于其他库格式。
