Abstract
Proteins often interact with each other to form complexes and play functional roles in almost all cellular processes. The study of protein–protein interactions is therefore critical to understand protein function and biological pathways. Affinity Purification coupled with Mass Spectrometry (AP-MS) is an invaluable technique for identifying the interaction partners in protein complexes. In this approach, the protein of interest is fused to an affinity tag, followed by the expression and purification of the fusion protein. The affinity-purified sample is then analyzed by mass spectrometry to identify the interaction partners of the bait proteins. In this chapter, we detail the protocol for tandem affinity purification (TAP) based on the use of the FLAG (a fusion tag with peptide sequence DYKDDDDK) and hemagglutinin (HA) peptide epitopes. The immunoprecipitation using dual-affinity tags offers the advantage of increasing the specificity of the purification with lower nonspecific-background interactions.
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Acknowledgments
The authors would like to acknowledge the Higher Education Center of Excellence (HICoE) Grant (JJ-2021-004) awarded by the Ministry of Higher Education of Malaysia to TYL.
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Low, T.Y., Lee, P.Y. (2023). Tandem Affinity Purification (TAP) of Interacting Prey Proteins with FLAG- and HA-Tagged Bait Proteins. In: Mukhtar, S. (eds) Protein-Protein Interactions. Methods in Molecular Biology, vol 2690. Humana, New York, NY. https://doi.org/10.1007/978-1-0716-3327-4_6
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DOI: https://doi.org/10.1007/978-1-0716-3327-4_6
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Publisher Name: Humana, New York, NY
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Online ISBN: 978-1-0716-3327-4
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