Biology Reference
In-Depth Information
Add the rest of the supernatants to separate aliquots of the prepared anti-FLAG af-
finity gel. If necessary, add some more CoIP buffer for a total volume of 1 ml and
incubate with gentle shaking at 4
C overnight.
The next day, wash the anti-FLAG affinity gel at least 3
(ideally 6
) with
300
l CoIP buffer each. After the final washing step, remove all liquid carefully
without disturbing the gel pellet; add 20
m
Laemmli buffer, mix gently, heat
to 95
C for 5 min, and briefly spin it down. Take all of the supernatant carefully
without transferring any of the gel; add DTT to a final concentration of 200 mM
to all samples.
The samples, together with the cell lysates kept at
m
lof2
20
C, can now be analyzed
by SDS-PAGE and Western blot. Blots are blocked in 5% dried milk/TBST and then
incubated with the primary antibodies (anti-FLAG 1:4000 and anti-GFP 1:1000, re-
spectively) in blocking solution with gentle shaking at 4
C overnight. The following
day, blots are washed 3
with TBST and then incubated with secondary antibody
(anti-rabbit HRP 1:5000) for 1 h at room temperature. The blots are washed 3
with
TBST and 2
with TBS before adding ECL substrate according to the manufac-
turer's recommendation. Blots can be developed and bands visualized by standard
X-ray film exposure or using a luminescent image analyzer (such as LAS-1000
and with the following settings: exposure time from 30 s to 30 min, aperture 0.85,
and integration of 2
2 pixels).
The expected protein sizes of TAS2Rs are
40 kDa for FLAG-tagged receptors
and 67 kDa for receptor-GFP
2
fusion proteins. However, multiple bands can often be
observed, most likely due to differently glycosylated receptor proteins (
Reichling,
Meyerhof, & Behrens, 2008
). The expected protein sizes of TAS1Rs are
90 kDa
for FLAG-tagged receptors and 120 kDa for receptor-GFP
2
fusion proteins. How-
ever, the actual observed protein sizes can be greater as protein glycosylation has
also been observed for TAS1Rs (
Max et al., 2001
).
FLAG-tagged receptors and receptor-GFP
2
fusion proteins should be detectable
in all lysates of cells expressing the respective receptor constructs. In immunopre-
cipitates of cells expressing only one receptor or the mix control, however, only
FLAG-tagged receptors should be stained, a result that demonstrates the specificity
of the antibodies and the suitability of the protocol. On the other hand, a positive
CoIP result is obtained if receptor-GFP
2
fusion proteins can be detected in
immunoprecipitates of cells coexpressing this receptor construct together with a
FLAG-tagged receptor, a result that is indicative of an assembly of the two respective
receptor proteins through protein-protein interactions.
13.2.4
Bioluminescence resonance energy transfer
In the second experimental approach to investigate receptor oligomerization,
BRET experiments are carried out (
Fig. 13.3
). We used the BRET
2
technology
(PerkinElmer) with the luciferase of
Renilla reniformis
(Rluc) as BRET donor
and a modification of the green fluorescent protein (GFP
2
) as BRET acceptor. Both
proteins are fused to the C-terminus of the TAS1R and TAS2R receptor proteins.
