摘要：在多分子复合物中，蛋白执行细胞功能。同一蛋白参与不同的复合物执行不同的功能。当前，胞内蛋白互作识别方法不能够揭示体内结合的突变部位。单分子pull-down (SiMPull)实验把传统的pull-down 实验和荧光显微技术结合起来，呈现出胞内单一蛋白复合物的直观图像。
对蛋白相互作用进行分析，是了解细胞功能和调控的关键。Taekjip Ha及其同事建立了以单分子分辨率来解析来自细胞和组织的蛋白复合物的身份及化学计量特征的一种新方法。被称为“单分子下拉”（SiMPull）的这种方法能区分一种蛋白的多种关联状态，同时还允许通过“光漂白”层次分析来确定复杂的化学计量特征。该分析方法的潜力在各种不同场景下都得到了演示，其中包括 利用来自组织提取物、细胞器及膜蛋白的内生蛋白所进行的分析工作。
pull-down试验路线图
Proteins perform most cellular functions in macromolecular complexes. The same protein often participates in different complexes to exhibit diverse functionality. Current ensemble approaches of identifying cellular protein interactions cannot reveal physiological permutations of these interactions. Here we describe a single-molecule pull-down (SiMPull) assay that combines the principles of a conventional pull-down assay with single-molecule fluorescence microscopy and enables direct visualization of individual cellular protein complexes. SiMPull can reveal how many proteins and of which kinds are present in the in vivo complex, as we show using protein kinase A. We then demonstrate a wide applicability to various signalling proteins found in the cytosol, membrane and cellular organelles, and to endogenous protein complexes from animal tissue extracts. The pulled-down proteins are functional and are used, without further processing, for single-molecule biochemical studies. SiMPull should provide a rapid, sensitive and robust platform for analysing protein assemblies in biological pathways.
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