Real-time single-molecule coimmunoprecipitation of weak protein-protein interactions
Real-time single-molecule coimmunoprecipitation of weak protein-protein interactions
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DOI：
10.1038/nprot.2013.116
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发表时间：
2013-10-01
期刊：
影响因子：
14.8
通讯作者：
Yoon, Tae-Young
中科院分区：
文献类型：
--
作者：
Lee, Hong-Won;Ryu, Ji Young;Yoon, Tae-Young
免疫共沉淀（co-IP）分析是研究蛋白质-蛋白质相互作用的有用方法。目前，它涉及电泳和蛋白质印迹，这不是最佳的检测弱和短暂的相互作用。在这个协议中，我们描述了一个先进的版本的co-IP分析，使用实时，单分子荧光成像作为其检测方案。诱饵蛋白被拉下到全内反射（TIR）显微镜的成像平面上。与未纯化的细胞或组织提取物保持在反应室中，我们观察单一的蛋白质-蛋白质之间的相互作用的表面固定的诱饵和荧光蛋白标记的猎物蛋白在真实的时间。这种直接记录提供了co-IP分析的时间分辨率的五个数量级的改进。与单分子的灵敏度和毫秒级的时间分辨率，区分我们的方法从其他方法用于测量弱蛋白质-蛋白质相互作用，它是可能的，以量化的相互作用动力学和活性分数的天然，未标记的诱饵蛋白。实时单分子co-IP分析，从裂解物制备到动力学分析需要近似4小时才能完成，为揭示靶蛋白-蛋白相互作用的丰富动力学图像提供了一种通用途径，例如，它可以用于研究在蛋白-蛋白相互作用水平上驱动个体癌症的分子病变。
Coimmunoprecipitation (co-IP) analysis is a useful method for studying protein-protein interactions. It currently involves electrophoresis and western blotting, which are not optimized for detecting weak and transient interactions. In this protocol we describe an advanced version of co-IP analysis that uses real-time, single-molecule fluorescence imaging as its detection scheme. Bait proteins are pulled down onto the imaging plane of a total internal reflection (TIR) microscope. With unpurified cells or tissue extracts kept in reaction chambers, we observe single protein-protein interactions between the surface-immobilized bait and the fluorescent protein-labeled prey proteins in real time. Such direct recording provides an improvement of five orders of magnitude in the time resolution of co-IP analysis. With the single-molecule sensitivity and millisecond time resolution, which distinguish our method from other methods for measuring weak protein-protein interactions, it is possible to quantify the interaction kinetics and active fraction of native, unlabeled bait proteins. Real-time single-molecule co-IP analysis, which takes similar to 4 h to complete from lysate preparation to kinetic analysis, provides a general avenue for revealing the rich kinetic picture of target protein-protein interactions, and it can be used, for example, to investigate the molecular lesions that drive individual cancers at the level of protein-protein interactions.