Biology Reference
In-Depth Information
Table 8.1
Summary of the Different BRET Methods
Spectral Properties
(Emission, nm)
Method
Donor
Acceptor
Donor
Acceptor
Substrate
BRET
1
Rluc
EYFP
480
530
Coelenterazine h
Extended
BRET
Rluc
EYFP
480
530
EnduRen
BRET
2
DeepBlueC
TM
/
coelenterazine
400a
Rluc
GFP2
395
510
DeepBlueC
TM
/
coelenterazine
400a
Enhanced
BRET
2
Rluc8
GFP2
395
510
BRET
3
Firefly
DsRed
565
583
D
-Luciferin and
cofactors
BRET
3
Rluc/
Rluc8
mOrange
480
564
EnduRen/
coelenterazine h
QD-BRET
Rluc
QD
480
605
Coelenterazine h
Over the last years, different generations of BRET (BRET
1
,BRET
2
, eBRET
2
, BRET
3
, and QD-BRET) have
been developed, depending on the type of enzyme substrate and the nature of donor/acceptor pairs. As
a result, the nomenclature for describing each of the BRET forms has not followed a unique rigorous
pattern.
interest should be considered. With the development of molecular dynamic
simulation of receptor-receptor interactions, it is nowadays possible to predict
the receptor interface. Interesting interaction hot spot(s) can be identified and
mutated to serve as negative control(s) (
Borroto-Escuela, Romero-Fernandez,
et al., 2010
).
3. Selection of the cell system and coexpression of the two BRET fusion proteins at a
relevant physiological level of expression
. Transient transfection (e.g., calcium
phosphate, Lipofectamine, and FuGENE HD) or virus-based transfection
systems have been used successfully. Common mammalian cells (e.g.,
HEK293T, COS-7, and CHO) and primary neuronal culture cells are compatible
with BRET assays. Depending on the type of BRET assay, different ratios of
donor/acceptor should be used/tested (see Points 1 in
Sections 3.1 and 3.2
). For
some assays, we may consider the use of bicistronic vectors, which may
guarantee a more homogeneous receptor ratio expression level.
4. Detection of the BRET signal and design of an appropriate assay
. The BRET
signal can be measured from adherent cells, cell suspensions, subcellular
fractions, purified proteins, and also culture medium in the case of secreted
proteins using a white plate to avoid light absorption and in parallel a black plate
to record total fluorescence values. The biological material (50-100
m
l) is then
dispatched into the 96-well plate and 10-20
m
l substrate is added (5
m
M final
