Bartoschik Tanja, Gupta Amit, Kern Beate, Hitchcock Andrew, Adams Nathan B P, Tschammer Nuska
NanoTemper Technologies GmbH, Munich, Germany.
Department of Molecular Biology and Biotechnology, University of Sheffield, Sheffield, UK.
Methods Mol Biol. 2020;2168:51-62. doi: 10.1007/978-1-0716-0724-4_2.
The combination of MicroScale Thermophoresis (MST) and near-native site-specific His-tag labeling enables simple, robust, and reliable determination of the binding affinity between proteins and ligands. To demonstrate its applicability for periplasmic proteins, we provide a detailed protocol for determination of the binding affinity of phosphite to three ABC transporter periplasmic-binding proteins from environmental microorganisms. ABC transporters are central to many important biomedical phenomena, including resistance of cancers and pathogenic microbes to drugs. The protocol described here can be used to quantify protein-ligand and protein-protein interactions for other soluble, membrane-associated and integral membrane proteins.
微量热泳技术（MST）与近天然位点特异性组氨酸标签标记相结合，能够简单、稳健且可靠地测定蛋白质与配体之间的结合亲和力。为证明其对周质蛋白的适用性，我们提供了一份详细方案，用于测定亚磷酸盐与三种来自环境微生物的ABC转运蛋白周质结合蛋白的结合亲和力。ABC转运蛋白在许多重要的生物医学现象中起着核心作用，包括癌症和致病微生物对药物的抗性。此处所述的方案可用于量化其他可溶性、膜相关和整合膜蛋白的蛋白质-配体以及蛋白质-蛋白质相互作用。
