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low expression, but we found that receptor constructs showing expression levels be-
low 35% of the mean expression level will not be suitable for performing CoIP and
BRET experiments.
13.2.3 Coimmunoprecipitation
In a first experimental approach to investigate receptor complexes, CoIP experi-
ments are performed ( Fig. 13.2 ). Two different receptors (or the same receptor with
two different tags or marker protein moieties) are coexpressed. Then, immunopre-
cipitation of one receptor is performed using a specific antibody against this receptor
or its tag. Subsequently, the precipitates are analyzed by Western blots for the pres-
ence of the originally immunoprecipitated receptor and for the presence of the other
receptor.
This type of approach had already been performed for in vitro analysis of
TAS1Rs ( Nelson et al., 2002 ). In these experiments, the authors used one tagged re-
ceptor (with HA-tag) and one unmodified receptor as a receptor-specific antibody
FIGURE 13.2
Detection of physical interactions between taste receptors by coimmunoprecipitation (CoIP).
(A) Receptors with a C-terminal FLAG-tag or fusion with GFP 2 , respectively, are coexpressed
in a mammalian cell line, for example, HEK293T cells. Cells are then lysed; membrane
proteins are solubilized and subjected to an immunoprecipitation with anti-FLAG affinity
beads. These immunoprecipitates are then separated by SDS-PAGE and analyzed for the
presence of thedifferent receptors byWesternblots using anti-FLAGandanti-GFPantisera. (B)
Sample Western blots of a CoIP experiment with TAS2R44 and TAS2R5. The receptor-GFP 2
protein can only be immunoprecipitated when coexpressed with a FLAG-tagged receptor,
which shows physical interactions between the two analyzed receptors. 1,
immunoprecipitation (IP) with lysates from cells expressing only TAS2R44-FLAG; 2, IP
with lysates from cells expressing TAS2R5-GFP 2 ; 3, IP with cells coexpressing of TAS2R44-
FLAG and TAS2R5-GFP 2 ; 4, mixed lysates of cells expressing either TAS2R44-FLAG or
TAS2R5-GFP 2 . IP always with anti-FLAG; upper blot: anti-FLAG, lower blot: anti-GFP.
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