Beck David B, Bonasio Roberto
Department of Medicine, Columbia University Medical Center, New York, New York.
Epigenetics Program, University of Pennsylvania Perelman School of Medicine, Philadelphia, Pennsylvania.
Curr Protoc Chem Biol. 2017 Jun 19;9(2):128-146. doi: 10.1002/cpch.18.
Identification of molecular interactions is paramount to understanding how cells function. Most available technologies rely on co-purification of a protein of interest and its binding partners. Therefore, they are limited in their ability to detect low-affinity interactions and cannot be applied to proteins that localize to difficult-to-solubilize cellular compartments. In vivo proximity labeling (IPL) overcomes these obstacles by covalently tagging proteins and RNAs based on their proximity in vivo to a protein of interest. In IPL, a heterobifunctional probe comprising a photoactivatable moiety and biotin is recruited by a monomeric streptavidin tag fused to a protein of interest. Following UV irradiation, candidate interacting proteins and RNAs are covalently biotinylated with tight spatial and temporal control and subsequently recovered using biotin as an affinity handle. Here, we describe experimental protocols to discover novel protein-protein and protein-RNA interactions using IPL. © 2017 by John Wiley & Sons, Inc.
识别分子间相互作用对于理解细胞如何发挥功能至关重要。大多数现有技术依赖于对目标蛋白及其结合伴侣进行共纯化。因此，它们在检测低亲和力相互作用方面能力有限，并且不能应用于定位于难以溶解的细胞区室的蛋白质。体内邻近标记（IPL）通过基于蛋白质在体内与目标蛋白的接近程度对蛋白质和RNA进行共价标记，克服了这些障碍。在IPL中，一种包含光可激活部分和生物素的异双功能探针被融合到目标蛋白上的单体链霉亲和素标签招募。紫外线照射后，候选相互作用蛋白和RNA在严格的空间和时间控制下被共价生物素化，随后使用生物素作为亲和手柄进行回收。在这里，我们描述了使用IPL发现新型蛋白质-蛋白质和蛋白质-RNA相互作用的实验方案。© 2017约翰威立国际出版公司
