Mechanisms of Olfaction in Insects - ResearchSpace@Auckland ...

Functional characterisation of Epiphyas postvittana odorant receptor 1 42
and left on ice for 30 minutes. The mixture was heated at 37°C until phase separation
occurred and the aqueous phase was removed completely. The remaining lower phase
after removal of the aqueous phase was the pre-condensed Triton X-114 and this was
diluted to 2% (in PBS) for use in membrane extraction.
For extraction of the membrane proteins, the transfected Sf9 cells were washed twice
with ice cold PBS. The cells were then dislodged in 400 µL PBS, spun down at 3000g
for three minutes and the pellet resuspended in 200 µL Lysis buffer (2% pre-
condensed Triton X-114 made up in PBS). The cells were sonicated (2x 30 second
passes), incubated on ice for at least one hour and then spun at 13000g at 4°C for ten
minutes to remove insoluble debris. The supernatant was then transferred to a new
tube, incubated at 37°C for ten minutes, followed by centrifugation at 13000g at 25ºC
for ten minutes to separate phases. The aqueous phase was discarded and the bottom
layer was mixed with 1 mL of fresh cold PBS and incubated at 0°C for ten minutes.
The mixture was heated at 37°C for ten minutes to separate phases and centrifuged at
13000g at 25ºC for ten minutes. The aqueous phase was again discarded and the
washing steps repeated twice. The protein was resuspended in 25 µL of the following
mix for running on a western blot: 6.25 µL of NuPage LDS Buffer, 2.5 µL of NuPage
sample reducing agent, 16.25 µL of distil water and 6 M urea. It was then heated at
37°C for 20 minutes and spun at 13000g for 30 seconds to remove any particulates.
Ten microlitres was loaded on a NuPage 4–12% Bis-Tris gel (Invitrogen) and run at
200 volts for 35 minutes. The iBlot Dry Blotting System (Invitrogen) was used to
transfer protein from the gel to a nitrocellulose transfer membrane following
manufacturer‟s instructions. 20 mL of 1x milk powder in 1x PBS with 0.05% Tween
20 was used for blocking the membrane for two hours. The blocking buffer was
washed off with 1x PBS 0.05% Tween 20 and the membrane incubated with a one in
1000 dilution in 1x PBS with 0.05% Tween 20 with the primary antibody, anti-myc
mouse antibody (Roche) for one and half hours. The antibody solution was washed
off and the membrane washed three times with 1x PBS containing 0.05% Tween 20.
The membrane was then incubated with secondary antibody, goat-anti mouse IgGiAP
(Stressgen, diluted one in 2000) for one hour. The secondary antibody solution was
washed off and 1-Step NBT-BCIP (Nitro blue tetrazolium chloride 5-Bromo-4-
chloro-3-indolyl phosphate, toluidine salt, Pierce) substrate was added to visualise
bands on the membrane.