Probing 2-dimensional protein-protein interactions on model membranes
Probing 2-dimensional protein-protein interactions on model membranes
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DOI：
10.1038/nprot.2006.270
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发表时间：
2006-01-01
期刊：
影响因子：
14.8
通讯作者：
Piehler, Jacob
中科院分区：
文献类型：
--
作者：
Gavutis, Martynas;Lata, Suman;Piehler, Jacob
该方案描述了一种用于测量膜锚定蛋白之间相互作用的动力学和亲和力的体外方法。这种方法是为剖析细胞因子与其受体亚基的相互作用动力学而特别建立的。为此，受体亚基通过使用多价络合脂类以定向的方式连接到固体支撑的脂质双层上。结合表面敏感光谱检测技术，探讨了配体与受体亚基之间的相互作用。通过反射干涉法的无标记检测用于随后的膜组装和受体亚基的定量连接。全内反射光谱用于监测配体与膜锚定受体的结合，通过FRET监测配体与受体的相互作用，以及监测配体交换动力学。这些方法可用于测定膜平面内配体-受体复合体的亲和力和稳定性。该方案中描述的技术可以在2-3个月内建立。
This protocol describes an in vitro approach for measuring the kinetics and affinities of interactions between membrane-anchored proteins. This method is particularly established for dissecting the interaction dynamics of cytokines with their receptor subunits. For this purpose, the receptor subunits are tethered in an orientated manner onto solid-supported lipid bilayers by using multivalent chelator lipids. Interaction between the ligand with the receptor subunits was probed by a combination of surface-sensitive spectroscopic detection techniques. Label-free detection by reflectance interferometry is used for following assembly of the membrane and tethering of the receptor subunits in quantitative terms. Total internal reflection spectroscopy is used for monitoring ligand binding to the membrane-anchored receptor, for monitoring ligand-receptor interactions by FRET and for monitoring ligand-exchange kinetics. These assays can be used for determining the affinities and stabilities of ligand-receptor complexes in plane of the membrane. The techniques described in this protocol can be established in 2-3 months.