Cattoglio Claudia, Pustova Iryna, Darzacq Xavier, Tjian Robert, Hansen Anders S
Department of Molecular and Cell Biology, University of California, Berkeley, Berkeley, CA, USA.
Li Ka Shing Center for Biomedical and Health Sciences, Berkeley, CA, USA.
Bio Protoc. 2020 Feb 20;10(4):e3526. doi: 10.21769/BioProtoc.3526.
Protein-protein interactions constitute the molecular foundations of virtually all biological processes. Co-immunoprecipitation (CoIP) experiments are probably the most widely used method to probe both heterotypic and homotypic protein-protein interactions. Recent advances in super-resolution microscopy have revealed that several nuclear proteins such as transcription factors are spatially distributed into local high-concentration clusters in mammalian cells, suggesting that many nuclear proteins self-interact. These observations have further underscored the need for orthogonal biochemical approaches for testing if self-association occurs, and if so, what the mechanisms are. Here, we describe a CoIP protocol specifically optimized to test self-association of endogenously tagged nuclear proteins (self-CoIP), and to evaluate the role of nucleic acids in such self-interaction. This protocol has proven reliable and robust in our hands, and it can be used to test both homotypic and heterotypic (CoIP) protein-protein interactions.
蛋白质-蛋白质相互作用构成了几乎所有生物过程的分子基础。免疫共沉淀（CoIP）实验可能是用于探测异型和同型蛋白质-蛋白质相互作用最广泛使用的方法。超分辨率显微镜的最新进展表明，诸如转录因子等几种核蛋白在哺乳动物细胞中呈空间分布于局部高浓度簇中，这表明许多核蛋白会自我相互作用。这些观察结果进一步强调了需要采用正交生化方法来测试是否发生自缔合，如果发生，其机制是什么。在这里，我们描述了一种经过专门优化的CoIP方案，用于测试内源性标记的核蛋白的自缔合（自CoIP），并评估核酸在这种自我相互作用中的作用。在我们手中，该方案已被证明是可靠且稳健的，并且可用于测试同型和异型（CoIP）蛋白质-蛋白质相互作用。
