BRET Principle
BRET is based on the fact that the energy derived from a luciferase reaction can be used to excite a fluorescent protein if the latter is in close proximity to the luciferase enzyme. The technology involves fusion of donor (luciferase) and acceptor (fluorescent) molecules to proteins of interest. Co-expression of fusion constructs in living cells enables their interaction to be studied in real time in a quantitative manner. Energy is transferred through nonradiative dipole–dipole coupling from the donor to the acceptor when in close proximity (typically, within 10 nm), resulting in fluorescence emission at a characteristic wavelength. The energy emitted by the acceptor relative to that emitted by the donor is termed the BRET signal, or BRET ratio. It is dependent upon the spectral properties, ratio, distance and relative orientation of the donor and acceptor molecules, as well as the strength and stability of the interaction between the proteins of interest.
BRET is very similar to FRET, but in FRET also the donor is a fluorescent protein that has to be excited. As BRET does not require external light source to excite the donor, it has very low background and does not suffer from issues often associated with FRET, such as autofluorescence, light scattering, or photobleaching. Other advantages of BRET are that it is a non-radioactive and homogeneous technology, and that the ratiometric signal minimises interferences from assay conditions.
BRET methods
Over the last years different BRET methods have been developed. All of them have their limitations and benefits. Various donor and acceptor pairs and their corresponding wavelengths can be found in the table below:
| Method | Donor | Substrate | Donor Emission [nm] | Acceptor | Acceptor Emission [nm] |
|---|---|---|---|---|---|
| BRET 1 | RLuc | Coelenterazine | 480 | eYFP | 530 |
| BRET 2 | RLuc | Coelenterazine 400a (Deep Blue C™) | 395 | GFP | 510 |
| eBRET 2 | RLuc8 | Coelenterazine 400a (Deep Blue C™) | 395 | GFP | 510 |
| BRET 3 | Firefly | Luciferin | 565 | DsRed | 583 |
| QD-BRET | RLuc/RLuc8 | Coelenterazine | 480 | QDot | 605 |
| NanoBRET | NanoLuc® | Furimazine | 460 | HaloTag® Ligand | 618 |
BRET 1
The original BRET method using Coelenterazine as substrate is nowadays called BRET 1. It is characterized by strong signals and long life-time.
BRET 2
In comparison with BRET 1, it has better separated donor and acceptor emission peaks. This makes BRET 2 a better choice for screening assays where high signal to noise ratios are required. A clear limitation of BRET 2 is the low light emission and the short life-time.
Enhanced BRET 2 (eBRET)
eBRET leads to approximately 5-fold better signal as in the original BRET 2 version. eBRET uses a new Renilla luciferase mutant, Rluc8.
BRET 3
The Firefly luciferase in BRET 3 shows lower cellular autofluorescence at the emission wavelength (565 nm) but disadvantages are weak signals and overlap between donor and acceptor emission peaks.
QD-BRET
A brand-new BRET version is the Quantum Dot-BRET (QD-BRET). The emission peaks are clearly separated which makes QD-BRET ideal for screening applications. Disadvantages are the large size of the QD molecules (1.5 - 6 nm) and the fact that genetical coding of QD-proteins is not possible. QD proteins cannot be expressed in living cells but must be added.
NanoBRET™
NanoBRET™ uses a luciferase much brighter than conventional luciferases, NanoLuc®, and has very good separation between donor emission (460 nm) and acceptor emission (618 nm).
Microplate Readers recommended for BRET
A microplate reader or luminometer capable of measuring luminescence with high-sensitivity using filters is the best combination for BRET; readers with monochromators can also be used, but with reduced sensitivity. It is necessary to perform 2 measurements at different wavelengths, and hence a reader with at least 2 emission filters is required (this is not possible, for example, in readers using one single filter cube with 1 excitation filter + 1 emission filter). All microplate readers below are recommended for BRET.
