Pull-down assay to identify proteins interacting with the small protein AtpΘ (Norf1) in Synechocystis sp. PCC 6803

Small proteins in prokaryotes are usually defined as polypeptides that are shorter than 80 amino acids. In the model cyanobacterium Synechocystis sp. PCC 6803 (Synechocystis 6803), the existence of several small proteins was verified previously. Among them, Norf1 (renamed to AtpΘ afterwards) is a small protein that is highly accumulated in darkness or stationary phase. To clarify the functions of AtpΘ, co-immunoprecipitation was performed to identify its interaction partners using FLAG-tagged AtpΘ protein as bait and FLAG-tagged sfGFP as negative control in three biological replicates each.

原核生物中的小型蛋白通常被定义为氨基酸残基数少于80的多肽链。在模式蓝细菌集胞藻（Synechocystis sp. PCC 6803，以下简称集胞藻6803）中，此前已有研究证实存在多种小型蛋白。其中，Norf1（后续更名为AtpΘ）是一种在黑暗环境或稳定生长期中高度富集的小型蛋白。为阐明AtpΘ的生物学功能，本研究以FLAG标签标记的AtpΘ蛋白为诱饵，以FLAG标签标记的sfGFP作为阴性对照，每组均设置三次生物学重复，通过免疫共沉淀（co-immunoprecipitation）实验鉴定其互作蛋白伙伴。