FRETBRET

FRETBRET is a term used in biophysics and cell biology to describe an integrated energy-transfer assay that combines elements of FRET (Förster resonance energy transfer) and BRET (bioluminescence resonance energy transfer). The approach uses a bioluminescent donor for the BRET pathway and a fluorescent donor for the FRET pathway, enabling two complementary readouts from a single biological interaction in living cells.

In FRETBRET experiments, a luciferase-based donor (for BRET) emits light that can excite a fluorescent acceptor via energy transfer, while a fluorescent donor-acceptor pair provides a separate FRET readout when optically excited. This dual-pathway design allows simultaneous monitoring of molecular proximity and conformational changes, offering distinct readouts from the same biological event.

Applications include mapping protein–protein interactions, monitoring receptor activation, and developing multiplexed biosensors; it is particularly useful for distinguishing signal from autofluorescence or photobleaching. FRETBRET can enhance the interpretability of results by providing corroborating signals from two energy-transfer mechanisms.

Advantages include a dual readout that increases confidence and reduces artifacts; the BRET component minimizes the need for external illumination, enabling improved suitability for live-cell imaging. Limitations include increased experimental complexity, potential spectral overlap between donors and acceptors, the need for careful calibration, and higher reagent and instrument demands.

History and use of FRETBRET vary across laboratories. The concept has appeared in the literature as a method to integrate FRET and BRET approaches, but its adoption is not as widespread as standard FRET or BRET assays, and protocols continue to evolve. Related techniques remain FRET and BRET as standalone methods.