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Using Tripartite Split-sfGFP for the Study of Membrane Protein–Protein Interactions

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Arabidopsis Protocols

Part of the book series: Methods in Molecular Biology ((MIMB,volume 2200))

Abstract

The study of protein–protein interaction (PPI) is critical for understanding cellular processes within biological systems. The conventional biomolecular fluorescence complementation (BiFC) or bipartite split-fluorescent protein (FP) is a noninvasive fluorescent-based technique that enables direct visualization of PPI in living cells once the two nonfluorescent fragments are brought into close vicinity. However, BiFC can potentially lead to a high background noise arising from an inherent feature of the irreversible self-assembly of the nonfluorescent fragments. Recently, the newly developed tripartite split-sfGFP method was demonstrated to detect membrane PPIs in plant cells without spurious background signals even when fusion proteins are highly expressed and accessible to the compartments of interaction. Here we describe a protocol for using the ß-Estradiol-inducible tripartite split-sfGFP assay for side-by-side analyses of in vivo PPI along with in situ subcellular localization of fusion proteins in agroinfiltrated Nicotiana benthamiana leaves.

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Acknowledgments

This work was supported by grants from the Ministry of Science and Technology of the Republic of China (MOST 103-2311-B-007-012-MY2 and MOST 105-2621-M-007-001-MY3). I also appreciate Ms. Chang-Yi Chiu’s help with the proof-reading and for some comments on the manuscript.

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Authors and Affiliations

  1. Department of Life Science and Institute of Bioinformatics and Structural Biology, National Tsing Hua University, Hsinchu, Taiwan

    Tzu-Yin Liu

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  1. Tzu-Yin Liu
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Correspondence to Tzu-Yin Liu .

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Editors and Affiliations

  1. Centro Nacional de Biotecnología, CSIC Centro Nacional de Biotecnología, Madrid, Spain

    Jose J. Sanchez-Serrano

  2. Centro de Investigaciones Biologicas, CSIC Madrid Centro de Investigaciones Biologicas, Madrid, Spain

    Julio Salinas

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Liu, TY. (2021). Using Tripartite Split-sfGFP for the Study of Membrane Protein–Protein Interactions. In: Sanchez-Serrano, J.J., Salinas, J. (eds) Arabidopsis Protocols . Methods in Molecular Biology, vol 2200. Humana, New York, NY. https://doi.org/10.1007/978-1-0716-0880-7_15

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  • DOI: https://doi.org/10.1007/978-1-0716-0880-7_15

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  • Publisher Name: Humana, New York, NY

  • Print ISBN: 978-1-0716-0879-1

  • Online ISBN: 978-1-0716-0880-7

  • eBook Packages: Springer Protocols

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