Stroth Nikolas
Center for Molecular Medicine, Karolinska Institute and University Hospital, Stockholm, Sweden.
J Biol Methods. 2016 Jan 28;3(1):e36. doi: 10.14440/jbm.2016.97. eCollection 2016.
The present protocol describes a method by which interactions between G protein-coupled receptors (GPCR) and intracellular proteins can be monitored in real-time and without the use of exogenous labels. The method is based on surface plasmon resonance (SPR) and uses synthetic peptides as mimics of intracellular GPCR domains. These peptides are covalently immobilized onto sensor chips and brought into contact with putative interacting proteins in the flow cells of the SPR instrument. The method allows flexible experimental designs, rapid testing of hypotheses and quantitative analysis of interactions. Relative to other established methods, it provides both an alternative and a complementary approach with several key advantages. The present protocol describes the method step-by-step, using the interaction between the serotonin 5-HT receptor and the calcium-binding protein S100B as an example.
本方案描述了一种方法，通过该方法可以实时监测G蛋白偶联受体（GPCR）与细胞内蛋白质之间的相互作用，且无需使用外源标记。该方法基于表面等离子体共振（SPR），并使用合成肽作为细胞内GPCR结构域的模拟物。这些肽被共价固定在传感器芯片上，并与SPR仪器流通池中的假定相互作用蛋白接触。该方法允许灵活的实验设计、快速的假设检验和相互作用的定量分析。相对于其他已确立的方法，它提供了一种具有几个关键优势的替代和互补方法。本方案以血清素5-HT受体与钙结合蛋白S100B之间的相互作用为例，逐步描述了该方法。
