Key Words: GFP, confocal, fluorescence, fluorophore, bioluminescence, FRET
Definition:BRET (similar to FRET) is a fluorescence technique, used to observe intermolecular interactions, which depends on the transfer of energy from one fluorophore (the donor) to another fluorophore (the acceptor). In BRET the donor is bioluminescent while the acceptor is fluorescent
TECHNOLOGY:
In BRET energy transfer reactions, the donor for energy transfer is a luminescent molecule (excited by the enzyme Renilla luciferase) and not a fluorescent molecule. The BRET acceptor is usually a GFP. Use of an enzyme to excite the donor molecule avoids the use of excitation light, and can therefore help to eliminate background autofluorescence in living cells. As in FRET studies, overlap between the emission spectra from the donor and the acceptor can reduce signal resolution.
APPLICATIONS:
BRET (like FRET) can be used to monitor molecular interactions in live cell imaging applications. BRET has been used, for example, to study GPCRs by investigating the association of receptor oligomerisation.
MICROSCOPE CONFIGURATION:
Any Nikon inverted epi-fluorescence microscope can be configured to carry out BRET studies. Widefield techniques can be compromised by emissions originating from above and below the plane of focus. These can be eliminated using confocal imaging (EC1, C1, C1si,). Spectral confocal imaging has particular advantages for BRET studies in that closely related spectra can be easily and cleanly separated, and quantitative BRET analysis is possible with spectral unmixing and ratio calculation.
RECOMMENDED SYSTEM:
The Eclipse Ti-E with the C1si spectral confocal system.