Department of Biology, University of Fribourg, Fribourg, Switzerland.
Methods Mol Biol. 2023;2704:173-181. doi: 10.1007/978-1-0716-3385-4_10.
Determining the affinity and specificity of protein-lipid interactions is crucial for understanding the physiological function and mode of action of signaling lipids, including steroids. Here we describe a method that relies on microscale thermophoresis (MST) to monitor the binding of sterols and steroids to proteins. The protein of interest is expressed as a polyhistidine-tagged fusion in E. coli and purified by affinity chromatography on a nickel-based resin. The purified protein is then labeled with a fluorescent dye and incubated with a serial dilution of the lipid ligand. The protein-ligand interaction is monitored by MST, which detects the fraction of the protein bound to the ligand based on its altered mobility in a thermal gradient. A binding curve fitted to the measured data points is then used to calculate the corresponding dissociation constant.
确定蛋白质-脂质相互作用的亲和力和特异性对于理解信号脂质（包括类固醇）的生理功能和作用模式至关重要。在这里，我们描述了一种依赖于微尺度热泳动（MST）监测固醇和类固醇与蛋白质结合的方法。感兴趣的蛋白质在大肠杆菌中作为多组氨酸标记融合蛋白表达，并通过镍基树脂亲和层析进行纯化。然后，将纯化的蛋白质用荧光染料标记，并与脂质配体的一系列稀释液孵育。通过 MST 监测蛋白质-配体相互作用，根据其在热梯度中的迁移率改变来检测与配体结合的蛋白质部分。然后，将拟合测量数据点的结合曲线用于计算相应的解离常数。
