Biology Reference
In-Depth Information
927-40000), TBS buffer (100 mM Tris/HCl pH 7.5; 1.5 mM NaCl), Tween 20,
primary antibody, and secondary antibody; if secondary antibody coupled to
horseradish peroxidase (HRP) is used—substrate for HRP, proper detection
equipment—for example, Kodak Image Station 440CF from Kodak for HRP-
coupled antibodies and Odyssey from LI-COR Biosciences for infrared dye-
coupled antibodies
17.2
METHODS
17.2.1
Overexpression of GPCRs in mammalian cell lines
Taken together the low expression level of GPCRs in native tissue and the toughness
to raise antibodies to a lot of GPCRs, studying protein-protein interactions is mainly
performed upon overexpression of the GPCRs of interest in mammalian cell lines.
We perform coimmunoprecipitation (co-IP) and membrane coimmunoprecipitation
(mco-IP) assays in mammalian cell lines transiently or stably overexpressing a
tagged form (HA, FLAG, c-myc, etc.) of the GPCR. Different cell lines can be used:
HEK293, CHO, HelaH21, COS, etc. The choice will mainly depend on the ease to
obtain a high transfection efficiency. HEK293 cells are very easy to work with and a
transfection efficiency of more than 95% can be achieved.
Different transfection methods including polyethylenimine (PEI), Ca
3
(PO
4
)
2
,
and lipofection can be used for this cell line (protocols in
Box 17.1
), all with their
advantages and disadvantages (
Table 17.1
). Forty-eight hours posttransfection, the
cells are collected in PBS and centrifuged for 10 min (250 g, 4
C). The cell pellets
can be stored at
70
C for several weeks.
17.2.2
Cell lysis
A lot of different cell lysis methods were developed, including mechanical disruption
and detergent-based methods, which we can subdivide in denaturing and nondena-
turing methods. Next to this, combinations of different methods can be performed to
achieve an optimal result. Each experiment has different requirements, causing the
need to optimize the lysis method for each particular experiment.
Procedures for GPCR immunoprecipitation experiments were optimized not only
to efficiently separate the hydrophobic membrane proteins from the hydrophilic pro-
teins but also to preserve the possible protein-protein interactions.
17.2.2.1
Mechanical disruption
Collected cells are resuspended in 1 ml 20 mM Tris/HCl pH 8.0 and 1 mM EDTA
and complemented with inhibitors (
Table 17.3
). This solution is sonicated at 20%
amplitude for 10 cycles; each cycle consists of 1 s on and 5 s off. This sonication
step disrupts the cell membrane and causes the release of the cellular content. When
these samples are centrifuged (20 min at 42,000
g
), a separation between
