Rich Joshua A, Gurung Sadeechya, Coates-Park Sasha, Liu Yueqin, Govil Anshika, Stetler-Stevenson William G, Peeney David
Laboratory of Pathology, Center for Cancer Research, National Cancer Institute, Bethesda, MD 20892, USA.
Laboratory of Pathology, Center for Cancer Research, National Cancer Institute, Bethesda, MD 20892, USA.
STAR Protoc. 2024 Dec 20;5(4):103509. doi: 10.1016/j.xpro.2024.103509. Epub 2024 Dec 12.
Biotin ligase-based proximity ligation is a widely used, highly effective technique for the study of in vivo protein-protein interactions. However, there are few reports and little consensus on the most effective methods for studying the proximal interactomes of secreted factors. Here, we present a protocol for studying extracellular proximal interactomes using an adaptation of TurboID/BioID2-based proximity ligation. We describe steps for cell preparation, sample collection, and initial processing. We then detail procedures for biotinylated protein enrichment, on-bead digestion, and post-pull-down processing. For complete details on the use and execution of this protocol, please refer to Peeney et al..
基于生物素连接酶的邻近连接是一种广泛应用于体内蛋白质-蛋白质相互作用研究的高效技术。然而，关于研究分泌因子近端相互作用组的最有效方法的报道较少，也缺乏共识。在此，我们提出了一种使用基于TurboID/BioID2的邻近连接改进方法来研究细胞外近端相互作用组的方案。我们描述了细胞制备、样本收集和初始处理的步骤。然后详细说明了生物素化蛋白富集、磁珠上消化和下拉后处理的程序。有关此方案的使用和执行的完整详细信息，请参考Peeney等人的文章。
