Biology Reference
In-Depth Information
the protein of interest from a lysate extracted from cell lines, primary cells, or native
tissue. The most critical step in this technique is cell lysis and solubilization of the
GPCRs; detergents are needed for extraction of GPCRs from the cell membrane, but
this process can lead to aggregation of the receptors, due to the hydrophobic nature of
the
a
-helices of the GPCR or to disruption of protein-protein contacts.
After polyacrylamide gel electrophoresis and Western blotting, the members of
the complex can be identified by specific antibodies.
In this chapter, we discuss general methods for carrying out and analyzing im-
munoprecipitation experiments and discuss the controls that should be included in
all valid immunoprecipitation experiments.
A number of GPCRs need posttranslational modifications to be properly
expressed and exert their normal function at the plasma membrane. N-glycosylation
is a common posttranslational modification of GPCRs. During biogenesis, glycosy-
lated GPCRs undergo a cycle of sugar chain additions and deletions by oligosacchar-
yltransferases and glucosidases. This is with the binding of calnexin and calreticulin
until the receptor obtains the correct folding and can leave the ER for further trans-
port to the Golgi where the glycosylation tree is further processed. The immature
ER-retained receptor can often be visualized on Western blot as a band with a lower
molecular weight. Therefore, detection of immature glycosylated GPCRs upon
coimmunoprecipitation can indicate that the protein-protein interaction starts already
in the ER (
Van Craenenbroeck, 2012; Van Craenenbroeck et al., 2011
).
17.1
MATERIALS
a. Cell culture (sterile)
: Dulbecco's modified Eagle's medium (DMEM),
penicillin-streptomycin, fetal calf serum (FCS),
L
-glutamine, EDTA-trypsin
(3 mM EDTA; 0.25% trypsin in PBS pH 7.4), phosphate-buffered saline (PBS),
cell culture plates, laminar flow cabinet, CO
2
incubator, and sterile pipettes
b. Transfection (sterile)
: transfection reagents (for details, see further) and plasmid
constructs encoding proteins of interest
c. Cell lysis
: lysis buffer (for details, see further), protease, and phosphatase
inhibitors (aprotinin,
b
-glycerophosphate, pefabloc, leupeptin), fridge or cold
room, microcentrifuge, and sonicator
d. Coimmunoprecipitation
: antibody (2
m
g/sample), Protein A or G beads, SDS
sample buffer, and heat block
e. SDS-PAGE and Western blot
: acrylamide/bisacrylamide, sodium dodecyl sulfate
(SDS), ammonium persulfate (APS), tetramethylethylenediamine (TEMED),
1.5 M Tris/HCl pH 8.8, 0.5 M Tris/HCl pH 6.8, electrophoresis buffer (49 mM
Tris; 0.38 M glycine; 0.1% SDS), transfer buffer (47 mM Tris; 37 mM glycine;
0.03% SDS; 20% MeOH), nitrocellulose membrane, Whatman paper,
polyacrylamide gel electrophoresis, and Western blot equipment (Bio-Rad)
f. Immunodetection
: nonfat dry milk or bovine serum albumin (BSA) or commercial
blocking buffer (e.g., Odyssey blocking buffer from LI-COR Biosciences, cat. nr.
