Biology Reference
In-Depth Information
by lipofection works equally well as transfection with calcium phosphate but is easier
to perform for many samples. The ratio of the plasmid DNAs should be three parts
receptor-GFP
2
and one part receptor-Rluc; this ratio has been optimized to yield
high BRET values (due to higher saturation of the BRET donors with BRET accep-
tors) while still resulting in high enough absolute light emission values to produce
reliable and reproducible results. For replicate measurements, cells in 2-3
wells should be transfected with the same receptor combination. An equal number
of wells with mock-transfected cells should be included as blanks. Also, cells trans-
fected with only the receptor-Rluc construct alone should be included as negative
control; cells transfected with the BRET vector serve as a positive control.
The next day, cells are washed twice with C1 buffer using an automated cell
washer leaving a volume of 50
l on the cells after the last wash. Also, before the
last wash, the bottom of the plates is covered with white tape to improve lumines-
cence detection. The plates are then read in a luminometer, which is able to perform a
simultaneous reading with two different channels (e.g., FLUOstar OPTIMA). For
reading the BRET assay, 20
m
M final con-
centration) is added to one well and light emission is detected simultaneously at 370-
450 and 500-530 nm, respectively, for 4-12 s before the instrument moves on to the
next well.
To analyze the data, the BRET ratio is calculated as light emission at 500-530 nm
(minus the light emission at 500-530 nm of mock-transfected cells) over light
emission at 370-450 nm (minus the light emission at 370-450 nm of mock-
transfected cells). The negative control of the receptor-Rluc transfected alone is
expected to give a BRET ratio of
m
l of 17.5
m
MDeepBlueC in C1 buffer (5
m
0.05, which is due to some residual light emission
of the coelenterazine substrate at 500-530 nm. The positive control, cells expressing
the direct fusion protein of GFP
2
-Rluc, will yield a very high BRET ratio, approach-
ing a value of 1. This is due to very high expression levels of this cytosolic protein
and, secondly, as a direct fusion protein, it provides all BRET acceptor moieties with
a BRET donor partner, leading to saturation and maximum BRET values. BRET ra-
tios of the receptor combinations will be much lower; they are expected to be in the
range of 0.05 to
0.4. Only values significantly higher than the values from the re-
spective receptor-Rluc alone are considered positive signals, that is, they stem from
receptors in close proximity to each other and are indicative of receptor-receptor
interactions.
We further recommend to always analyzing both possible receptor combinations,
for example, TAS2R16-GFP
2
with TAS2R44-Rluc and TAS2R16-Rluc with
TAS2R44-GFP
2
. The results may vary from each other. Possible explanations might
be differences in expression levels between the receptors; a higher BRET ratio will
be obtained when the receptor expressed at a higher level is fused to the BRET ac-
ceptor. Alternatively or additionally, the BRET donor and acceptor protein moieties
might be folded differently under the respective receptors, bringing them closer to-
gether or further apart in the different combinations. For these reasons, we generally
assumed that two receptors can interact with each other when at least one of the two
combinations yielded a positive BRET signal.
