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Protocol for establishing a protein-protein interaction network using tandem affinity purification followed by mass spectrometry in mammalian cells

Weixiang Bian,Hua Jiang,Shan Feng,Junjie Chen,Wenqi Wang,Xu Li

Summary Identification of protein interactors is fundamental to understanding their functions. Here, we describe a modified protocol for tandem affinity purification coupled with mass spectrometry (TAP/MS), which includes two-step purification. We detail the S-, 2×FLAG-, and Streptavidin-Binding Peptide (SBP)- tandem tags (SFB-tag) system for protein purification. This protocol can be used to identify protein interactors and establish a high-confidence protein–protein interaction network based on computational models. This is particularly useful for identifying bona fide interacting proteins for subsequent functional studies. For complete details on the use and execution of this protocol, please refer to Bian et al. (2021) .