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Design and implementation of bimolecular fluorescence complementation (BiFC) assays for the visualization of protein interactions in living cells

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Abstract

Bimolecular fluorescence complementation (BiFC) analysis enables direct visualization of protein interactions in living cells. The BiFC assay is based on the discoveries that two non-fluorescent fragments of a fluorescent protein can form a fluorescent complex and that the association of the fragments can be facilitated when they are fused to two proteins that interact with each other. BiFC must be confirmed by parallel analysis of proteins in which the interaction interface has been mutated. It is not necessary for the interaction partners to juxtapose the fragments within a specific distance of each other because they can associate when they are tethered to a complex with flexible linkers. It is also not necessary for the interaction partners to form a complex with a long half-life or a high occupancy since the fragments can associate in a transient complex and un-associated fusion proteins do not interfere with detection of the complex. Many interactions can be visualized when the fusion proteins are expressed at levels comparable to their endogenous counterparts. The BiFC assay has been used for the visualization of interactions between many types of proteins in different subcellular locations and in different cell types and organisms. It is technically straightforward and can be performed using a regular fluorescence microscope and standard molecular biology and cell culture reagents.

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Figure 1: Schematic diagram representing the principle of the BiFC assay.
Figure 2: Combinations of fusion proteins to be tested for bimolecular fluorescence complementation.
Figure 3: Determination of the specificity of bimolecular fluorescence complementation by mutational analysis.

Change history

  • 30 November 2006

    In the version of this article initially published online, the article’s page numbers should have been 1278–1286. In addition, the numbered items in Step 2 did not correspond to the format of the HTML version. In Steps 2–13, the locations of the TROUBLESHOOTING headings did not correspond to the Troubleshooting table. These errors have been corrected in the PDF version of the article.

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Acknowledgements

I thank C.-D. Hu for his participation in the design and implementation of the BiFC assay in mammalian cells and all members of the Kerppola laboratory for their contributions to the improvement and adaptation of the BiFC approach.

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Kerppola, T. Design and implementation of bimolecular fluorescence complementation (BiFC) assays for the visualization of protein interactions in living cells. Nat Protoc 1, 1278–1286 (2006). https://doi.org/10.1038/nprot.2006.201

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