FRET two-hybrid assay by linearly fitting FRET efficiency to concentration ratio between acceptor and donor

We here introduce a fluorescence resonance energy transfer (FRET) two-hybrid assay method to measure the maximal donor(D)- and acceptor(A)-centric FRET efficiency (ED,max and EA,max) of the D-A complex and its stoichiometry by linearly fitting the donor-centric FRET efficiency (ED) to the acceptor-to-donor concentration ratio (RC) and acceptor-centric FRET efficiency (EA) to 1/RC, respectively. We performed this method on a wide-field fluorescence microscope for living HepG2 cells co-expressing FRET tandem constructs and free donor/acceptor and obtained correct ED, EA, and stoichiometry values of those tandem constructs. Evaluation on the binding of Bad with Bcl-XL in Hela cells showed that Bad interacted strongly with Bcl-XL to form a Bad-Bcl-XL complex on mitochondria, and one Bad interacted mainly with one Bcl-XL molecule in healthy cells, while with multiple (maybe 2) Bcl-XL molecules in apoptotic cells.