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Updated: Nov 11, 2025

Identification of Protein Interacting Partners Using Tandem Affinity Purification
Published on: February 25, 2012
Functional proteomics protocol for the identification of interaction partners in Tetrahymena thermophila
Syed Nabeel-Shah1,2,3, Jyoti Garg1,4, Pata-Eting Kougnassoukou Tchara5,6
1Department of Chemistry and Biology, Ryerson University, 350 Victoria Street, Toronto, ON M5B 2K3, Canada.
Abstract:
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 Tetrahymena thermophila. 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).
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