1× electroblotting transfer buffe, Polyacrylamide gel containing proteins of interest, 100% methanol, Powder-free gloves, Test tube, Electro blotting apparatus, PVDF transfer membrane, Fiber pad, Whatman no. 1 filter paper, Trays, Magnetic Stirrer, Power supply.
Wet PVDF with Methanol for a few seconds.
Then transfer the PVDF membrane from the methanol to a dish containing 1x electroblotting transfer buffer and keeps for 5 minutes.
Dip two pieces of filter paper in 1x Electroblotting transfer buffer (above the PVDF membrane).
Soak the fiber pads in blotting buffer.
Remove gel for the electrophoresis cell and soak in Electroblotting buffer (above the fiber pad) for 15 minutes.
Then assemble the transblott unit.
For that Place the opened gel holder cassette on a clean, flat surface. Place one transfer buffer–wetted fiber pad on the top surface, followed by the filter paper.
Place the gel face down on the support, so that the cut edge is now on the right-handside.
Remove wet PVDF membrane from the tray containing transfer buffer and place above the gel. Roll a test tube across the membrane to avoid air bubbles between membrane and gel.
Place the other wetted filter paper above the PVDF membrane.
Place the other fiber pad on top of the membrane and close the holder.
Slide the assembled transfer cassette into the tank with the gel on the cathode side and the membrane on the anode side.
Place the Electroblotting apparatus above the magnetic stirrer. Drop the stir bar.
Fill the transfer tank with 1× transfer buffer so that the buffer completely covers theElectrode panels but does not touch the electrical connectors.
Turn on the Magnetic stirrer. Assemble the sandwich and electroblot at either constant voltage of 50Volts at room temp. for 30 minutes or constant current of 500 mA for the same amount of time.
At the end of the transfer period, turn off the power and disconnect the power supply. Open the transfer sandwich and remove the membrane and gel. Thoroughly rinse the membrane with high-purity water, three times, 5 min each.
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