Department of Molecular Biosciences, University of Texas at Austin, Austin, TX, USA.
Methods Mol Biol. 2022;2438:59-81. doi: 10.1007/978-1-0716-2035-9_4.
Mapping how proteins form complexes and change binding partners is central to understanding cell signaling. Bulk biochemistry can provide a summary of what complexes are present in a cell, but information about the diversity of individual protein complexes is lost. Here, we describe single-cell , single-molecule pull-down (sc-SiMPull), a TIRF microscopy-based coimmunoprecipitation method, to visualize thousands of individual proteins, their binding partners, and protein complex stoichiometry directly from single-cell lysate. By iterating sc-SiMPull over time, temporal dynamics of protein complexes in response to signaling can be constructed.
绘制蛋白质形成复合物以及改变结合伴侣的图谱对于理解细胞信号转导至关重要。批量生物化学可以提供细胞中存在的复合物的概述，但会丢失关于单个蛋白质复合物多样性的信息。在这里，我们描述了单细胞、单分子下拉（sc-SiMPull），这是一种基于全内反射荧光显微镜的共免疫沉淀方法，可直接从单细胞裂解物中可视化数千种单个蛋白质、它们的结合伴侣以及蛋白质复合物的化学计量。通过随时间迭代 sc-SiMPull，可以构建响应信号转导的蛋白质复合物的时间动态。
