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Increased Transfer Efficiency Using a Discontinuous Buffer System With the Trans-Blot SD Cell

ffer:
20 ml 5x Tris-CAPS 20 ml 5x Tris-CAPS
15 ml MEOH 1 ml 10 % SDS
65 ml water 79 ml water

Following vertical electrophoresis:
1. Wet the PVDF membrane in 100% MEOH, then equilibrate in bottom/anode buffer for at least 30 minutes. To this buffer add one sheet of Extra Thick Blotting Paper.

2. Equilibrate the acrylamide gel in top/cathode buffer. Soak a second piece of Extra Thick Blotting Paper in this buffer.

3. Prepare for transfer by making the gel sandwich as follows.
To bottom platinum anode add
Extra thick filter paper in bottom/anode buffer
Equilibrated PVDF membrane
Equilibrated gel
Extra thick filter paper in top/cathode buffer

4. Secure top stainless steel cathode, cover, and run at constant current,1.5 mA per square centimeter of gel (for example, 120 mA for a small 8 x 10 cm gel) for 3060 minutes.


References
1. Gallagher, S. R., Winston, S. E., Fuller, S. A. and Hurrell, J. G. R., in Current Protocols in Molecular Biology (Ausubel, F. M., Brent, R., Kingston, R. E., Moore, D. D., Seidman, J. G., Smith, J. A., and Struhl, K., Eds), pp.10.8.110.8.16, Greene Publishing and Wiley Interscience, New York. (1992)

2. Hirano, H., J. Protein Chem. 8, 115130 (1989).

3. Jacobson G., in Protein Blotting (Dunbar, B. S., Ed.), pp.5370, IRL Press, New York (1994).

4. Kyhse-Anderson, J., J. Biochem. Biophys. Methods, 10, 203209 (1984).

.5. Lauriere, M., Anal. Biochem., 212, 206211 (1993).

6. Lissilour, S. and Godinot, C., BioTechniques, 9, 397401 (1990).

7.
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