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Visualization of ternary complexes in living cells by using a BiFC-based FRET assay

Abstract

Studies of protein interactions have increased our understanding and knowledge of biological processes. Assays that utilize fluorescent proteins, such as fluorescence resonance energy transfer (FRET) and bimolecular fluorescence complementation (BiFC), have enabled direct visualization of protein interactions in living cells. However, these assays are primarily suitable for a pair of interacting proteins, and methods to visualize and identify multiple protein complexes in vivo are very limited. This protocol describes the recently developed BiFC–FRET assay, which allows visualization of ternary complexes in living cells. We discuss how to design the BiFC–FRET assay on the basis of the validation of BiFC and FRET assays and how to perform transfection experiments for acquisition of fluorescent images for net FRET calculation. We also provide three methods for normalization of the FRET efficiency. The assay employs a two-chromophore and three-filter FRET setup and is applicable to epifluorescence microscopes. The entire protocol takes about 2–3 weeks to complete.

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Figure 1: Principle of the BiFC–FRET assay.
Figure 2: Schematic view of cloning vectors for BiFC–FRET analysis.
Figure 3: Schematic view of BiFC–FRET analysis.
Figure 4: Calculations for CoA and CoB and net FRET (nF).

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Acknowledgements

We thank Drs. David Piston and Atsushi Miyawaki for kindly providing the cDNAs for Cerulean and Venus, as well as the Hu Laboratory members for their helpful discussions. C.D.H. was supported by grants from the Purdue Cancer Center (NCI-P30CA23168), the Indiana Elks Inc., Walther Cancer Institute, National Science Foundation (0420634-MCB) and the American Heart Association (0655570Z).

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Correspondence to Chang-Deng Hu.

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Shyu, Y., Suarez, C. & Hu, CD. Visualization of ternary complexes in living cells by using a BiFC-based FRET assay. Nat Protoc 3, 1693–1702 (2008). https://doi.org/10.1038/nprot.2008.157

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