To avoid membrane contamination, wear gloves during all the steps of the protocol.
1. Prepare the transfer buffer (see Notes 1-3) and cool it to 4°C before the end of the electrophoretic run.
2. Cut to the dimension of the gel, two pieces of filter paper and one piece of nitrocellulose/gel (see Note 4).
3. After electrophoresis (see Note 5), wash the gel in distilled water and then equilibrate it in transfer buffer. The ideal time for 1.5-mm gels is 10 to 15 min (see Note 6).
4. Soak the nitrocellulose membrane for 15-20 min in transfer buffer. Also wet two Scotch-Brite® fiber pads, gel and filter papers in transfer buffer.
5. Assemble the "sandwich" for transfer in this order: fiber pad, filter paper, nitrocellulose, gel, filter paper, and fiber pad. Remove all air bubbles between membrane and gel and between paper and gel.
6. Put the blot sandwich in the gel holder and hold it firmly, to ensure a tight contact between gel and membrane.
7. Fill the cell with transfer buffer and place a stirring bar inside the transfer cell, so that the buffer is stirred during electrotransfer and temperature and conductivity are uniform during electrotransfer.
8. Place the gel holder in the transfer cell with the sandwich oriented as follows: ANODE/fiber pad, filter paper, nitrocellulose, gel, filter paper, fiber pad/CATHODE.
9. Carry out blotting at a constant current until it has reached a total of 1.5-2.0 A (see Note 7), refrigerating the buffer to 4°C (see Note 8) for gels of 16 x 18 cm (such as 2D gels) or at constant voltage (100 V) for 1 h for minigels (see Note 9).
10. After electrotransfer, disassemble the blotting apparatus and remove the nitrocellulose membrane. To mark the orientation of the membrane, cut away the lower right corner, corresponding to low Mr, high pH.
The membrane can be processed immediately for immunoblotting or can be air-dried and stored at -20°C, within parafilm sheets for extended periods (29).
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