Aggarwal Vasudha, Ha Taekjip
Center for Biophysics and Computational Biology, University of Illinois Urbana Champaign, Urbana, IL, USA.
Bioessays. 2014 Nov;36(11):1109-19. doi: 10.1002/bies.201400090. Epub 2014 Aug 29.
Macromolecular interactions play a central role in many biological processes. Protein-protein interactions have mostly been studied by co-immunoprecipitation, which cannot provide quantitative information on all possible molecular connections present in the complex. We will review a new approach that allows cellular proteins and biomolecular complexes to be studied in real-time at the single-molecule level. This technique is called single-molecule pull-down (SiMPull), because it integrates principles of conventional immunoprecipitation with the powerful single-molecule fluorescence microscopy. SiMPull is used to count how many of each protein is present in the physiological complexes found in cytosol and membranes. Concurrently, it serves as a single-molecule biochemical tool to perform functional studies on the pulled-down proteins. In this review, we will focus on the detailed methodology of SiMPull, its salient features and a wide range of biological applications in comparison with other biosensing tools.
大分子相互作用在许多生物过程中起着核心作用。蛋白质-蛋白质相互作用大多通过免疫共沉淀进行研究，而这种方法无法提供关于复合物中所有可能分子连接的定量信息。我们将综述一种新方法，该方法能够在单分子水平上对细胞蛋白质和生物分子复合物进行实时研究。这项技术称为单分子下拉技术（SiMPull），因为它将传统免疫沉淀的原理与强大的单分子荧光显微镜技术相结合。SiMPull用于计数在细胞质和细胞膜中发现的生理复合物中每种蛋白质的数量。同时，它作为一种单分子生化工具，用于对下拉的蛋白质进行功能研究。在本综述中，我们将重点介绍SiMPull的详细方法、其显著特点以及与其他生物传感工具相比的广泛生物学应用。
