受体
互补
G蛋白偶联受体
配体(生物化学)
化学
兴奋剂
突变体
融合蛋白
生物化学
生物
重组DNA
基因
出处
期刊:Methods in molecular biology
日期:2022-01-01
卷期号:: 139-153
被引量:3
标识
DOI:10.1007/978-1-0716-2473-9_10
摘要
NanoLuc Binary Technology (NanoBiT) was recently developed by Promega, based on a large NanoLuc fragment (LgBiT) and two small complementation tags, the low-affinity SmBiT tag and the high-affinity HiBiT tag. In recent studies, we applied NanoBiT to ligand-binding assays of some G protein-coupled receptors via genetic fusion of a secretory LgBiT (sLgBiT) to the extracellular N-terminus of the receptors and covalent attachment of the low-affinity SmBiT tag to an appropriate position of their peptide ligands. The NanoBiT-based homogenous ligand-receptor binding assay is convenient for use and suitable for both the wild-type and mutant receptors, representing a novel tool for interaction mechanism studies of these receptors with their ligands. In the present chapter, we provide detailed protocols for setting up the NanoBiT-based homogenous binding assay using growth hormone secretagogue receptor type 1a (GHSR1a) and its endogenous agonist and antagonist as a representative model system.
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