Akaki Kotaro, Mino Takashi, Takeuchi Osamu
Department of Medical Chemistry, Graduate School of Medicine, Kyoto University, Kyoto 606-8501, Japan.
Bio Protoc. 2022 Aug 5;12(15). doi: 10.21769/BioProtoc.4478.
Detecting protein-protein interactions (PPIs) is one of the most used approaches to reveal the molecular regulation of protein of interests (POIs). Immunoprecipitation of POIs followed by mass spectrometry or western blot analysis enables us to detect co-precipitated POI-binding proteins. However, some binding proteins are lost during cell lysis or immunoprecipitation if the protein binding affinity is weak. Crosslinking POI and its binding proteins stabilizes the PPI and increases the chance of detecting the interacting proteins. Here, we introduce the method of DSP (dithiobis(succinimidyl propionate))-mediated crosslinking, followed by tandem immunoprecipitation (FLAG and HA tags). The eluted proteins interacting with POI can be analyzed by mass spectrometry or western blotting. This method has the potential to be applied to various cytoplasmic proteins. Graphical abstract.
检测蛋白质-蛋白质相互作用（PPI）是揭示目标蛋白质（POI）分子调控机制最常用的方法之一。对POI进行免疫沉淀，随后进行质谱分析或蛋白质免疫印迹分析，可使我们检测到共沉淀的POI结合蛋白。然而，如果蛋白质结合亲和力较弱，一些结合蛋白会在细胞裂解或免疫沉淀过程中丢失。交联POI及其结合蛋白可稳定PPI，并增加检测相互作用蛋白的机会。在此，我们介绍二硫代双（琥珀酰亚胺丙酸酯）（DSP）介导的交联方法，随后进行串联免疫沉淀（FLAG和HA标签）。与POI相互作用的洗脱蛋白可通过质谱分析或蛋白质免疫印迹进行分析。该方法有应用于各种细胞质蛋白的潜力。图形摘要。
