Biology Reference
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had been generated for this receptor. For almost all TAS2Rs, however, commercial
receptor-specific antibodies are either not available or are not working reliably
(
Behrens et al., 2012
). Therefore, differently tagged or marked receptors will have
to be used. We recommend the usage of FLAG-tagged receptors and receptor-GFP
2
fusion proteins, respectively.
To perform the CoIP experiments, cells are seeded at
20% confluence in 10 cm
cell culture dishes coated with poly-
D
-lysine. The next day, cells are transfected with
DNA for the receptor combination to be analyzed (one receptor-FLAG, one recep-
tor-GFP
2
construct) in a 1:1 ratio or with either single receptor construct (performed
in duplicate) using a lipofection reagent according to the manufacturer's recommen-
dation. One set of the cells transfected with only one receptor construct serves as
control for several purposes: by analyzing the cell lysate, expression of the construct
can be confirmed. Second, by using the cell lysates for the immunoprecipitation pro-
cedure, one can show that the procedure was performed successfully and with spec-
ificity, that is, that only the FLAG-tagged receptor can be pulled down but not the
GFP
2
-marked receptor expressed on its own. Third, both parts together also help to
confirm the specificity of the antibodies used. The second set of cells transfected with
a single receptor construct will be mixed later during the cell lysis procedure. This will
serve as control to verify that the observed protein-protein interactions did not arise
artificially during the purification process but were already present inside the cells.
The following day, cells are prepared for the lysis procedure by putting the cell
culture dishes on ice. Cells are washed twice with 10 ml cold PBS before adding 1 ml
of ice-cold CoIP buffer; incubate for 5-10 min on ice. Cells and cell remnants should
detach easily and be transferred to a glass homogenizer; move the pestle up and down
several times to fully lyse the cells and solubilize the membrane proteins. At this step,
one set of lysates of cells transfected with a single receptor can be mixed to control
for occurrence of protein-protein interactions inside the cells vs. during the proce-
dure. To ensure that all remaining cell debris and membrane fragments are removed
from the solubilized proteins, ultracentrifugation at 100,000
g
and 4
C should be per-
formed for 90 min.
In the meantime, the anti-FLAG-agarose beads used for immunoprecipitation of
FLAG-tagged proteins can be prepared. Be careful to always keep them at 4
Coron
ice! Resuspend the anti-FLAG affinity gel well by pipetting up and down with a
yellow pipette tip whose tip has been cut off and take out the appropriate amount
(use 20
m
l of agarose bead suspension per sample, which equals 10
m
l of packed
gel). Add ice-cold CoIP buffer (volume
volume of packed gel), mix gently,
and centrifuge for 30 s at 8200
g
,4
C. Remove the liquid without disturbing the gel
pellet and repeat this washing step twice more. After the final washing step, add
an appropriate amount of ice-cold CoIP buffer and aliquot the washed anti-FLAG
affinity gel on ice into new 1.5 ml tubes.
After the ultracentrifugation is finished, take the supernatant of the cell lysates
and put them on ice. Transfer 20
m
l of the lysates to new tubes and freeze these sam-
ples; they will later be separated by SDS-PAGE and analyzed by Western blots and
serve as controls for expression and antibody specificity (see the preceding text).
ΒΌ
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