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