Electroblotting to PVDF

Procedure

Note: Wear gloves for all procedures.

Cut 10 x 10 cm square of PVDF membrane on a clean glass with a sharp and clean razor blade.
Prewet cut PVDF membrane with Methanol.
Soak membrane in transfer buffer for 5-15 minutes.
Soak gel in transfer buffer for 15-30 minutes.
Prepare a sandwich of soaked gel and PVDF between Whatman 3MM (or similar) paper and assemble the blotting cassette according to the list below.
Note: It is critically important to not capture air bubbles between the membrane and the gel. This will block transfer of proteins to the membrane.
Lay down a cassette with Black side down and one sponge in a Tupperware; flood the cassette with some transfer buffer and rub the air bubbles out of the sponge.
Lay a piece of wet 3MM paper on the sponge.
Lay the soaked gel on the paper; smooth out bubbles.
Lay the soaked PVDF membrane on the gel; smooth out bubbles.
Lay a piece of wet 3MM paper on the membrane.
Lay a wet sponge on the paper.
Close and clip the cassette.
Transfer the cassette to the blotting tank prefilled with transfer buffer.
Layers should be as follows: sponge, blotter paper, gel, PVDF membrane, blotter paper, sponge.
Electrotransfer the proteins towards the anode for one hour at 100 mA.
Note: These conditions are for a 0.75 mm thick gel for proteins in the range of 25-65 kDa. Larger or lower molecular weight proteins usually require longer transfer times. Other tricks include including some SDS in the transfer buffer or lowering the MeOH concentration. You will need longer times for thicker or higher concentration gels. For example, 1.0 mm thick gels (6-10%) or 12-15% gels, use 1.5-2 hours at 100 mA.
Disassemble the cassette and stain the PVDF membrane with Amido Black for one minute in Tupperware container. Do not allow membrane to dry out.
Destain the PVDF membrane in 1% acetic acid with repeated changes until the bands are clearly visible and the background is reduced.
Wash the membrane in MilliQ water for several minutes before airdrying between two sheets of 3MM paper.
Stain the gel with Coomassie Blue R-250 for one hour. (optional)
Destain the gel with Coomassie destain. (optional)

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