Biology Reference
In-Depth Information
Lysis Buffer and transfer in 1 mL capacity Potter homogenizer. After 20 up and
down regular and gentle moves of the pestle in the glass mortar, transfer the cell ho-
mogenates in 1.5 mL microtubes, and pellet the membranes by 20 min centrifugation
at 43,000 g and at 4
C. Resuspend the membrane pellet in 200
L of Tris Lysis
Buffer and quantify the protein levels (Bradford reagent) twice with 5
m
m
L of sample.
Aliquot the membrane preparations (200
m
g/aliquot), freeze in liquid nitrogen, and
80
C for further use.
store at
7.2.2.2
Solubilization and deglycosylation of the samples
In transfected cells, 5-HT
4
receptors exist in many glycosylated forms, resulting in
smear bands on SDS-PAGE electrophoresis. To circumvent this problem, we add a
deglycosylation step of the receptor preparation prior to gel electrophoresis.
Thaw and pellet 400
g of each sample by 20 min centrifugation at 43,000 g and
at 4
C. To solubilize the membrane proteins, resuspend the pellet in 200
m
L of sol-
ubilization buffer, and incubate 2 h on a rotating wheel in a cold room at 4
C. Then,
pellet the remaining cell fragments by 20 min centrifugation at 43,000 g at 4
C and
collect the supernatant. Adjust the EDTA concentration in the sample buffer to in-
crease it to 10 mM EDTA (deglycosylation buffer), add 2
m
L (1000 units) of
N-glycosidase F, and incubate the tubes overnight at 37
C. After adding 67
m
lof
Laemmli buffer 4
to the sample, load 25
m
g of proteins of each sample on an ac-
rylamide gel.
m
7.2.2.3
Western blot and detection of the 5-HT
4
receptor dimers
Load the samples on Tris/HCl gels (8% or 10% acrylamide/bisacrylamide) in dena-
turing conditions (SDS). To achieve a good separation of the different dimer bands,
use 20 cm glass plate systems (settings, stacking 1 h at 100 V and separation 5 h at
200 V). Transfer the proteins on nitrocellulose using wet electrophoretic system (set-
tings, 30 V, overnight at 4
C). After transfer, saturate the nitrocellulose membrane
for 1 h in TBST-milk, then rinse it with TBST and incubate overnight with the anti-
Myc antibody diluted 1/1000 in TBST-milk, under gentle agitation at 4
C. Wash the
membrane six times, 5 min, in TBST, and then incubate it for 1 h with the secondary
antibody (e.g., antimouse antibody conjugated with HRP, 1/4000) diluted in TBST-
milk, under gentle agitation at room temperature. Wash the membrane six times,
5 min, in TBST, and then reveal the bands using a chemiluminescent kit according
to manufacturer instructions (e.g., Pierce ECL Western Blotting Substrate).
Using this technique, we are capable to detect monomers and dimers form for
each 5-HT
4
receptor variant (
Fig. 7.2
A). By cotransfection of a “long” variant with
a “short” one, for example, variant (a) with variant (e), we can discriminate the dimer
formation of (a) and (e) protomers (
Fig. 7.2
B). 5-HT
4
receptor dimers can also be
detected using a receptor devoid of its C-terminal domain (
Fig. 7.2
C) and this trun-
cated receptor is capable to associate with (a) or (b) variant (
Fig. 7.2
D).
To resume, the key steps in this technique are (1) to start with a membrane prep-
aration of proteins, (2) to apply a deglycosylation step to the samples, and (3) to use
long-separation gels.
