Biology Reference
In-Depth Information
immobilized. Thanks to the high affinity of Proteins A and G for binding antibodies,
a protein complex containing the antibody, the protein of interest and its interacting
proteins can be isolated. The origin of the antibody that is used for IP (species of
animal in which IgG was produced) defines the choice of the beads. In general, Pro-
tein A beads are used to bind and purify IgG from human and rabbit, whereas Protein
G beads are recommended for isolation of IgG from human, mouse, and rat. There
are also immobilized Protein A/G beads available, which combine the IgG-binding
domain of both Protein A and Protein G, enabling purification of numerous IgG sub-
classes and species. When one of the potential interacting partners has a common tag
(e.g., FLAG and HA), then it is possible to use beads immediately coupled to the
specific antibody directed against the tag (FLAG-beads and HA-beads). This will
save time—generally 4 h incubation of lysate with such beads is sufficient to obtain
high immunoprecipitation efficiency. However, the immobilization can reduce the
antibody's affinity to the antigen and prevent IP.
Typically, co-IP is performed with total cell lysates, but when studying mem-
brane proteins, modifications of this technique can be recommended. We will first
describe a standard co-IP protocol and then a protocol to isolate specifically com-
plexes with plasma membrane expressed receptors. A schematic overview is given
in
Fig. 17.2
.
17.2.3.1
Standard coimmunoprecipitation: in total cell lysate
(Fig. 17.2A, left)
RIPA lysis seems to be one of the best choices to obtain the starting material for iso-
lating GPCRs together with its interacting proteins, as it is an efficient cell lysis
method resulting in clear bands on SDS-PAGE (
Fig. 17.1
). The cleared lysates
are transferred to clean microcentrifuge tubes: (1) a fraction (10%) of each sample
will be analyzed on SDS-PAGE to check protein expression (both expression level
and molecular weight will be evaluated) and (2) to the rest of the lysate 2
m
g of the
antibody is added and incubated for 2-4 h at 4
C upon rotation. Next, 20
m
l of pure
Protein A beads (delivered as a 50% slurry) is added per sample. These beads are first
washed three times (centrifugation after each washing step: 110
g
,4
C, 1 min)
with RIPA buffer supplemented with protease and phosphatase inhibitors (see par-
agraph about cell lysis). The washed beads are added to the lysates and incubated
overnight with rotation at 4
C.
Note
: Preclearing of the lysates can be performed to remove proteins from the
sample that can bind nonspecifically to the beads. To perform this step, lysates need
to be incubated with the beads (without addition of antibody) for 30-60 min at 4
C
with rotation. Then, samples are centrifuged for 1 min (3000
g
,4
C) and clear ly-
sates are transferred to clean microcentrifuge tubes. Such precleared samples can be
used for immunoprecipitation. This step is only necessary if “contaminating” pro-
teins interfere with visualization of the proteins of interest.
The next day, beads are again washed three times in RIPA buffer (supplemented
with protease and phosphatase inhibitors) to remove all proteins present in the lysate,
which are not bound. When nonspecific signals are observed after Western blot
