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Nat Protoc. 2008;3(11):1693-702. doi: 10.1038/nprot.2008.157.

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

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Department of Medicinal Chemistry and Molecular Pharmacology and Purdue Cancer Center, Purdue University, West Lafayette, Indiana 47907, USA.


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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