Biology Reference
In-Depth Information
these sequences to the 5
0
and 3
0
ends of the GFP
2
and Rluc sequences, respectively.
Next, these fragments are individually cloned into one cassette at the 3
0
end of one
receptor sequence; the correct sequence and the maintenance of the open reading frame
should be confirmed. Because of its small size, the FLAG-tag can be added to the re-
ceptor sequence directly by PCR, bypassing this first cloning step. Subsequently, using
restriction enzymes 2 and 3, the receptor sequence in the constructs in the preceding
text can be replaced with the sequences of all other receptors. Alternatively, restriction
enzyme 1 in combination with restriction enzyme 3 can be used to replace the complete
sequence of sst-TAS2R with that of another sst-TAS2R sequence or with the se-
quences of TAS1R2 and TAS1R3, respectively.
13.2.2
Expression analysis of receptor constructs
The finished receptor constructs should be analyzed for their expression—good
expression levels are needed for both the CoIP and BRET experiments. Receptor-
Rluc protein expression can be easily quantified via luminescence activity of the lu-
ciferase moiety; receptor-GFP
2
proteins can by quantified by GFP
2
fluorescence.
The constructs are transiently transfected into the desired mammalian cell line
(e.g., HEK293T cells) in 96-well plate format. Cells are seeded at
10% confluence
in white, clear-bottomed, poly-
D
-lysine-coated 96-well plates (for luminescence de-
tection) or black, clear-bottomed, poly-
D
-lysine-coated 96-well plates (for fluores-
cence detection) and transfected 48 h later using a lipofection reagent according
to the manufacturer's recommendation; the confluence of the cells should be
50% before transfection. For replicate measurements, cells of three wells should
be transfected with the same receptor construct. It is also advantageous for the anal-
ysis to include mock-transfected cells as negative control and cells transfected with
the BRET vector (expressing a direct fusion protein of GFP
2
-Rluc) as a positive
control.
The following day, cells are washed twice with C1 buffer using an automated cell
washer leaving a volume of 50
l on the cells. Washing the cells manually bears the
risk of cells detaching from the bottom of the plate. GFP
2
fluorescence can imme-
diately be detected in a fluorescence plate reader (such as FLIPR) using excitation at
488 nm (a suboptimal but satisfactory excitation wavelength; the ideal excitation
wavelength would be at 395 nm) and emission detection at 515 nm. For measuring
luciferase activity, the bottom of the plates is covered with white tape before the last
wash. The plates are then read in a luminometer (such as FLUOstar OPTIMA). In this
protocol, 20
m
M final concentration) is
added to one well, and light emission is detected at 370-450 nm before the instru-
ment moves on to the next well.
For both fluorescence and luminescence measurements, the obtained values for
the receptor constructs will be much lower compared to the BRET vector, which is to
be expected as the receptor fusion proteins are integral membrane proteins, whereas
the GFP
2
-Rluc fusion protein is a cytosolic protein and can therefore reach much
higher expression levels. It is difficult to suggest a general cutoff for unacceptably
m
l of 17.5
m
M DeepBlueC in C1 buffer (5
m
