Biology Reference
In-Depth Information
background BRET
2
ratio, which corresponds to the signal obtained with cells
expressing the BRET donor alone (see Point 5 in
Section 3
).
5.
In saturation assays, specific BRET
2
ratio values are plotted as a function of the
GFP2/Rluc fusion protein ratio. Therefore, the total amount of luminescence
(BRET donor amount) and fluorescence (BRET acceptor amount) must be
determined for each transfection. It is important that the BRET donor levels are
relatively constant throughout the experiment. In case of significant variation
(difference of 20% or more from the average value), the corresponding points
should be excluded from the final plot or the experiment repeated again. To
quantify the amount of BRET acceptor in each well, the fluorescence is measured
at 510 nm after external excitation at 410 nm in black 96-well microplates.
Background fluorescence is obtained by determining fluorescence in wells
containing untransfected cells (GFP2 zero (0) value) and subtracted from the
fluorescence values measured in cells expressing increasing amounts of BRET
acceptor (GFP2) to obtain the specific GFP2 values. Often when distributing
cell suspensions into white opaque 96-well microplates (see Point 2 in
Section 3.1
), a parallel and similar procedure is performed in black 96-well
micro
plates in order to quantify the amount of the acceptor by fluorescence
measurements. Otherwise, cells are detached from spare wells of the white 96-
well plate using 100
m
l PBS-EDTA or Versene, washed twice with PBS, and
collected by centrifugation for 5 min at 300
g
at RT. The pellet is resuspended
l PBS and transferred to a black 96-well plate for fluorescence
measurements.
6.
GFP2-GFP20/Rluc8 fusion protein ratio is calculated for each data point.
Depending on the application, it may be necessary to convert luminescence and
fluorescence values into absolute amounts of interacting partners using standard
curves correlating luminescence and fluorescence signals with amounts of
proteins (see Point 7 in
Section 3.1
).
7.
Correlations are assessed between fluorescence or luminescence values and
receptor expression levels in BRET
2
experiments. When appropriate
radioligands are available, determination of receptor expression levels in BRET
2
assays can be relevant to ascertain that the expression level of fusion proteins falls
within the physiological range. It can also be useful to determine the true
acceptor/donor ratio in BRET
2
saturation experiments. Luminescence and
fluorescence levels of several receptor-donors and receptor-acceptors are found
to be linearly correlated with receptor densities (
Borroto-Escuela, Garcia-
Negredo, Garriga, Fuxe, & Ciruela, 2010
). This correlation is an intrinsic
characteristic of each fusion protein, and therefore, correlation curves have to be
established for each construct. Cells are transfected (a reasonable number of
different independent transfections should be performed) with different
quantities of either the BRET donor or the BRET acceptor fusion protein
plasmid. Then, the luciferase activity (for the BRET donor receptor) and
fluorescence values (for the BRET acceptor receptor) are determined as
described earlier. In parallel, saturation radioligand binding experiments are
in 150-200
m
