Protocol for Quantitative Proteomics of Cellular Membranes and Membrane Rafts - LC-MS/MS in Proteomics: Methods and Applications - page 241

3.4.WesternBlotting
forFlotillin-1

1. Samples separated by SDS-PAGE are transferred onto
nitrocellulose membranes electrophoretically. These direc-
tions assume the use of the XCell 'Sure Lock' Blot Module
Kit (Invitrogen) and can transfer two gels at a time. This
can be adapted for use with other kits.
2. Two extra thick filter papers per gel are cut to size and
soaked in transfer buffer along with sponge pads for blot-
ting immediately prior to electrophoresis. Soak the nitro-
cellulose in water.
3. The gel unit is disconnected from the power supply unit
and disassembled. The stacking gel is discarded and the
resolving gel is then laid on top of the nitrocellulose mem-
brane. The nitrocellulose membrane is laid on top of one of
the wetted filter papers and the other wetted filter is placed
on top of the gel creating a filter paper-gel-nitrocellulose-
filter paper sandwich. Place two sponge pads in the cath-
ode core of the blot module. Place the filter paper-gel-
nitrocellulose-filter paper sandwich on top of the sponge
pads orientated such that the gel is closer to the cathode
than the nitrocellulose. Place enough pre-soaked sponge
pads on top of the assembly so that the assembly is of the
same depth as the cathode core. If transferring two gels,
place one sponge pad on top of the assembly then place
the second filter paper-gel-nitrocellulose-filter paper sand-
wich on top followed by more pre-soaked sponge pads as
required. Place the anode core on top of the assembly and
secure in place in the buffer tank.
4. Fill the inner blot module with transfer buffer and the outer
buffer chamber with water.
5. Place the lid on the tank and activate the power supply.
Transfer is accomplished by electrophoresis at 35 V con-
stant for 2 h.
6. Once transfer is complete, remove the blot module from
the buffer tank and carefully disassemble it. Remove the
nitrocellulose membrane and place it in a staining dish. The
pre-stained molecular weight markers should be clearly vis-
ible on the nitrocellulose.
7. Incubate membrane in 10 mL blocking buffer at room
temperature for 1 h on a rotary platform shaker.
8. Discard the blocking buffer and briefly rinse the mem-
brane in TBS-T prior to addition of a 1:2000 dilution of
flotillin-1 antibody prepared in TBS-T/2% BSA and left
overnight at 4 ◦ C on a rotary platform shaker.
9. Remove the primary antibody and wash the membrane
three times for 10 min each using 10 mL TBS-T.