Cautions in Measuring In Vivo Interactions Using FRET and BiFC in Nicotiana benthamiana

  • Meral Tunc-Ozdemir
  • Yan Fu
  • Alan M. JonesEmail author
Part of the Methods in Molecular Biology book series (MIMB, volume 1363)


Bimolecular fluorescence complementation (BiFC) and Förster Resonance Energy Transfer (FRET) are two widely used techniques to investigate protein–protein interactions and subcellular compartmentalization of proteins in complexes. As of January 2015, there were 805 publications retrieved by PUBMED with the query “bimolecular fluorescence complementation” and 11,327 publications retrieved with the query “fluorescence resonance energy transfer”. Only a few of these publications describe studies of plant cells. Given the importance and popularity of these techniques, applying them correctly is crucial but unfortunately many studies lack proper controls and verifications. We describe (1) BiFC and FRET problems that are frequently encountered at different stages of the protocols, (2) how to use appropriate controls, and (3) how to apply plant transformation and imaging procedures. We provide step-by-step protocols for the beginner to obtain high quality, artifact-free BiFC and FRET data.

Key words

Bimolecular fluorescence complementation (BiFC) Förster resonance energy transfer (FRET) Plant cells Confocal microscopy ImageJ Nicotiana benthamiana 



We thank Tony Perdue for training and critical comments on the manuscript and Nguyen Phan for training on BiFC and FRET imaging. Financial support in the Jones Lab is provided by the National Science Foundation and the National Institutes of General Medicine. The Division of Chemical Sciences, Geosciences, and Biosciences, Office of Basic Energy Sciences of the US Department of Energy through the grant DE-FG02-05er15671 to A.M.J. funds technical support in the Jones Lab.


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Copyright information

© Springer Science+Business Media New York 2016

Authors and Affiliations

  1. 1.Department of BiologyUniversity of North Carolina at Chapel HillChapel HillUSA
  2. 2.Department of PharmacologyUniversity of North Carolina at Chapel HillChapel HillUSA

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