Biology Reference
In-Depth Information
separation of two forms of the dopamine D
4
receptor. The lower band of around
46 kDa represents nonmature, ER-associated receptor, and a second band of
52 kDa represents mature receptor with the fully processed N-linked glycosylation
tree. For elution of proteins from the beads, presence of reducing agent seems to be
extremely important, as without addition of DTT, only a smear at the top of the blot
can be detected (aggregated proteins). After elution at 95
C, less mature receptor
can be isolated then after elution at nonboiling temperatures (37 or 50
C). The
strong band around 50 kDa on the upper, right panel represents immunoglobulin,
which was used for IP.
17.2.5
Detection of interacting partners
Finally, when the proteins are eluted from the beads, standard SDS-PAGE can be
performed to separate proteins according to their molecular weight. Next, proteins
are transferred from the polyacrylamide gel to a nitrocellulose or polyvinylidine
fluoride (PVDF) membrane during Western blot procedure and further immunode-
tection can be done with specific antibodies.
In coimmunoprecipitation studies often immunoreactive bands can be detected at
the molecular level corresponding to approximately twice the molecular weight of
monomeric receptors. This observation can already suggest that the receptor exists
in dimerized form. Such dimer is resistant to SDS denaturation and this might indi-
cate the involvement of hydrophobic interactions in dimerization (
Salahpour,
Angers, & Bouvier, 2000
).
If the elution of proteins was performed at 37
C, the IgG used for IP will appear
at the height of 100 kDa (association of two antibody heavy chains), and when 95
C
is used, the antibody band will be observed at 50 kDa. This has to be taken into ac-
count when the protein under investigation has similar molecular weight, for exam-
ple, a lot of class A GPCRs have a molecular weight around 50 kDa, and, therefore,
we recommend for immunodetection a secondary antibody that recognizes (1) only
the light chain of the primary antibody or (2) that is conformation-specific and can
recognize only nondenatured antibody (antibody used for IP is denatured and will not
be recognized). If primary antibody directly coupled to the enzyme (e.g., HRP) or
infrared dye is available, this can also be a good alternative. Next, primary antibodies
produced in another animal species than the antibody used for IP can facilitate the
interpretation of the Western blot immunodetection (see
Fig. 17.3
—IB with anti-HA
rabbit), but for close related species, the cross recognition may occur. All these pro-
cedures will help to clearly visualize the protein of interest, which otherwise would
be masked by immunoglobulin band.
17.2.6
Controls
To exclude aspecific binding of proteins to the beads or antibody, it is recommended
to include in the experimental setup a control sample to which no antibody or irrel-
evant antibody will be added during immunoprecipitation procedure. Moreover, to
