Abstract
Deciphering protein–protein interactions (PPIs) in vivo is crucial to understand protein function. Bimolecular fluorescence complementation (BiFC) makes applicable the analysis of PPIs in many different native contexts, including human live cells. It relies on the property of monomeric fluorescent proteins to be reconstituted from two separate subfragments upon spatial proximity. Candidate partners fused to such complementary subfragments can form a fluorescent protein complex upon interaction, allowing visualization of weak and transient PPIs. It can also be applied for investigation of distinct PPIs at the same time using a multicolor setup. In this chapter, we provide a detailed protocol for analyzing PPIs by doing BiFC in cultured cells. Proof-of-principle experiments rely on the complementation property between the N-terminal fragment of mVenus (designated VN173) and the C-terminal fragment of mCerulean (designated CC155) and the partnership between HOXA7 and PBX1 proteins. This protocol is compatible with any other fluorescent complementation pair fragments and any type of candidate interacting proteins.
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Acknowledgments
Research in the laboratory of S. Merabet is supported by Association pour la Recherche sur le Cancer (ARC, PJA20141202007); Fondation pour la Recherche Médicale (FRM, DEQ. 20170336732); Ligue Nationale Contre le Cancer, Centre National de Recherche Scientifique (CNRS); CEFIPRA (5503-2); CNRS; and Ecole Normale Supérieure (ENS) de Lyon. We further thank the China Scholarship Council (CSC, File No. 201708070003) for the doctoral grant to Y. Jia.
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Jia, Y., Bleicher, F., Reboulet, J., Merabet, S. (2021). Bimolecular Fluorescence Complementation (BiFC) and Multiplexed Imaging of Protein–Protein Interactions in Human Living Cells. In: Zamir, E. (eds) Multiplexed Imaging. Methods in Molecular Biology, vol 2350. Humana, New York, NY. https://doi.org/10.1007/978-1-0716-1593-5_12
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DOI: https://doi.org/10.1007/978-1-0716-1593-5_12
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