Husain Afzal, Begum Nasim A, Kobayashi Maki, Honjo Tasuku
Deptartment of Biochemistry, Faculty of Life Sciences, Aligarh Muslim University, Aligarh, India.
Department of Immunology and Genomic Medicine, Graduate School of Medicine, Kyoto University, Kyoto, Japan.
Bio Protoc. 2020 Dec 5;10(23):e3837. doi: 10.21769/BioProtoc.3837.
Protein-protein interactions play key roles in nuclear processes including transcription, replication, DNA damage repair, and recombination. Co-immunoprecipitation (Co-IP) followed by western blot or mass spectrometry is an invaluable approach to identify protein-protein interactions. One of the challenges in the Co-IP of a protein localized to nucleus is the extraction of nuclear proteins from sub-nuclear fractions without losing physiologically relevant protein interactions. Here we describe a protocol for native Co-IP, which was originally used to successfully identify previously known as well novel topoisomerase 1 (TOP1) interacting proteins. In this protocol, we first extracted nuclear proteins by sequentially increasing detergent and salt concentrations, the extracted fractions were then diluted, pooled, and used for Co-IP. This protocol can be used to identify protein-interactome of other chromatin-associated proteins in a variety of mammalian cells.
蛋白质-蛋白质相互作用在包括转录、复制、DNA损伤修复和重组在内的核过程中发挥着关键作用。免疫共沉淀（Co-IP）后进行蛋白质印迹或质谱分析是鉴定蛋白质-蛋白质相互作用的一种非常有价值的方法。将定位于细胞核的蛋白质进行免疫共沉淀时面临的挑战之一是从亚核组分中提取核蛋白，同时又不丢失生理相关的蛋白质相互作用。在此，我们描述了一种天然免疫共沉淀的方案，该方案最初用于成功鉴定先前已知的以及新的拓扑异构酶1（TOP1）相互作用蛋白。在本方案中，我们首先通过依次增加去污剂和盐的浓度来提取核蛋白，然后将提取的组分进行稀释、合并，并用于免疫共沉淀。该方案可用于鉴定多种哺乳动物细胞中其他染色质相关蛋白的蛋白质相互作用组。
