Department of Chemistry and Biology, Ryerson University, 350 Victoria Street, Toronto, ON M5B 2K3, Canada.
Donnelly Centre, University of Toronto, Toronto, ON, M5S 3E1, Canada.
STAR Protoc. 2021 Mar 4;2(1):100362. doi: 10.1016/j.xpro.2021.100362. eCollection 2021 Mar 19.
We describe an optimized protocol for one-step affinity purification of FZZ-tagged proteins followed by mass spectrometry analysis for the identification of protein-protein interactions in the ciliate protozoan . The FZZ epitope tag contains 2 protein A moieties (ZZ) and a 3xFLAG separated by a TEV cleavage site, which can also be employed in tandem affinity purification. This protocol is versatile and is suitable to use for other common epitope tags and can be adapted for other ciliates. For complete details on the use and execution of this protocol, please refer to Garg et al. (2019).
我们描述了一种优化的一步亲和纯化 FZZ 标记蛋白的方案，随后通过质谱分析鉴定纤毛虫原生动物中的蛋白质-蛋白质相互作用。FZZ 表位标签包含 2 个蛋白 A 结构域（ZZ）和一个通过 TEV 切割位点隔开的 3xFLAG，也可用于串联亲和纯化。该方案具有通用性，适用于其他常见的表位标签，并可适用于其他纤毛虫。有关该方案的使用和执行的完整详细信息，请参阅 Garg 等人。（2019 年）。
