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Visualization of protein interactions in living Caenorhabditis elegans using bimolecular fluorescence complementation analysis

Nature Protocols volume 3, pages 588–596 (2008)Cite this article

Abstract

The bimolecular fluorescence complementation (BiFC) assay is a powerful tool for visualizing and identifying protein interactions in living cells. This assay is based on the principle of protein-fragment complementation, using two nonfluorescent fragments derived from fluorescent proteins. When two fragments are brought together in living cells by tethering each to one of a pair of interacting proteins, fluorescence is restored. Here, we provide a protocol for a Venus-based BiFC assay to visualize protein interactions in the living nematode, Caenorhabditis elegans. We discuss how to design appropriate C. elegans BiFC cloning vectors to enable visualization of protein interactions using either inducible heat shock promoters or native promoters; transform the constructs into worms by microinjection; and analyze and interpret the resulting data. When expression of BiFC fusion proteins is induced by heat shock, the fluorescent signals can be visualized as early as 30 min after induction and last for 24 h in transgenic animals. The entire procedure takes 2–3 weeks to complete.

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Figure 1: Principle of the bimolecular fluorescence complementation assay.
Figure 2: Schematic view of worm bimolecular fluorescence complementation (BiFC) cloning vectors.

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Acknowledgements

We thank Dr. Atsushi Miyawaki for kindly providing cDNAs of Venus, and the members of the Hu laboratory for helpful discussions. R.E.E. was supported by a grant from the American Cancer Society. C.-D.H. was supported by the Purdue Cancer Center (NCI-P30CA23168), Indiana Elks, Walther Cancer Institute and grants from National Science Foundation (0420634-MCB) and American Heart Association (0655570Z).

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Authors and Affiliations

  1. Department of Medicinal Chemistry and Molecular Pharmacology, Purdue University, West Lafayette, 47907-2091, Indiana, USA

    Y John Shyu, Susan M Hiatt, Holli M Duren & Chang-Deng Hu

  2. Department of Molecular Biology, University of Medicine and Dentistry of New Jersey-School of Osteopathic Medicine, 2 Medical Center Drive, Stratford,, 08084, New Jersey, USA

    Ronald E Ellis

  3. Department of Biological Chemistry, Howard Hughes Medical Institute, University of Michigan Medical School, Ann Arbor, 48109-0650, Michigan, USA

    Tom K Kerppola

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Correspondence to Chang-Deng Hu.

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Shyu, Y., Hiatt, S., Duren, H. et al. Visualization of protein interactions in living Caenorhabditis elegans using bimolecular fluorescence complementation analysis. Nat Protoc 3, 588–596 (2008). https://doi.org/10.1038/nprot.2008.16

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