Quantification of Protein in Brain Tissue by Western Immunoblot Analysis - Drugs of Abuse: Neurological Reviews and Protocols - page 261

Fig. 1. Assembling the electroblotter: In a tray with transfer buffer, wet the fi ber
pad and the blotting paper. Prewet the PVDF membrane in methanol and put on top
of the blotting paper. Cut the stacking gel from the running gel with a gel spacer and
discard. Put the running gel on top of the PVDF membrane, add the second fi ber pad,
assemble the cassette, and slide into the transfer unit. When attaching the power cords,
make sure the red cord (positive cord) is on the side of the membrane, whereas the
black cord (negative cord) is on the side of the gel.
membrane with a soft pencil to keep track of the orientation ( see Fig. 1 ).
5. Place the gel on top of the PVDF membrane.
6. Add a second fi ber pad, close the cassette, and transfer to the electrophoresis unit
( see Notes 5 and 6 ). The gel should be on the side of the cathode and membrane
on the side of anode ( see Fig. 1 ).
7. Transfer for 2-3 h at 300 mA.
8. Disassemble transfer, discard the gel, and keep the membrane. Quickly rinse
the membrane under tap water to wash off residual gel pieces. Proceed either
to the antibody reaction, or store the membrane in blocking solution overnight
at 4°C, or let the membrane dry. If the membrane dries, it has to be rewetted
in 100% methanol.
3.4. Antibody Reaction (Fig. 2)
1. Place the wet membrane into blocking solution for 1 h at room temperature with
gentle shaking ( see Note 7 ).
2. Wash for 7-10 min in PBST at room temperature with gentle shaking.
3. Incubate in primary antibody, made up in PBST, for 2 h at room temperature
with gentle shaking.
4. Wash four times for 7-10 min in PBST at room temperature with gentle shaking.
5. Incubate in secondary antibody, made up in PBST, for 1 h at room temperature