C.B.S. Scientific EBU-402 - Manual (Page 10)

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Double & Triple-Wide Mini-Blotters Instruction Manual Oct 2012
SECTION 4
Applications & Running Conditions for Tank Type Electro-Blotting
4.1 Recommended Buffers, Power Settings, and Transfer Times
Precise transfer conditions will vary according to the number and type of gels used, buffer
conditions employed, power input, and the general goal of the experiment. Refer to the reference
section for in depth discussions on practical and theoretical approaches to gel transfers.
Using the enclosed Towbin buffer system use the following conditions at constant voltage:
For the EBU-302 and EBU-402 Blotting Systems:
At 4°C: 26V, current range: 250-300mAmp (depending on number and thickness of gels)
for 18 hours. At room temperature: 26V, current range: 400-500mAmp for 18 hours
For the EBU-362 Blotting Systems:
At 4°C: 60V, current range: 100-600mAmp (depending on number and thickness of gels)
for 18 hours. At room temperature: 26V, current range: 400-500mAmp for 18 hours
Towbin Buffer – 1X, 20% MeOH:
0.025M Tris Base
0.192 M Glycine
0.05-0.1%(w/v) SDS
20% (v/v) Methanol
4.2 References
1. Ausubel, F.M., Brent, R., Kingston, R.E., Moore, D.D., Seidman, J.G., Smith, J.A., Struhl, K.
(ed.) (1993). Current Protocols in Molecular Biology. Vol. 2, Greene Publishing Associates,
Inc. and John Wiley & Sons, Inc., Ch.10.
2. Burnette, W.N. (1981). Western blotting: Electrophoretic transfer of proteins from sodium
dodecyl sulfate-polyacrylamide gels to unmodified nitrocellulose and radiographic detection
with antibody and radioiodinated protein A. Anal. Biochem. 112:195-203.
3. Peluso, R.W. and Rosenberg, G.H. (1987). Quantitative electrotransfer of proteins from
sodium dodecyl sulfate polyacrylamide gels onto positively charged nylon membranes. Anal.
Biochem. 162:389-398.
4. Perides, G. Plagens, U., and Traub, P. (1986). Protein transfer from fixed, stained and dried
polyacrylamide gels and immunoblot with protein A-gold. Anal. Biochem. 152:94-99.
5. Tesfaigzi, J., Smith-Harrison, W., and Carlson, D.M. (1994). A simple method for reusing
western blots on PVDF membranes. Biotechniques. 17:268-269.
6. Towbin, H., Staehelin, T., and Gordon, J. (1979). Electrophoretic transfer of proteins from
polyacrylamide gels to nitrocellulose sheets: Procedure and some applications. Proc. Natl.
Acad. Sci. U.S.A. 76: 4350-4354.
7. Sambrook, J., Fritsch, E.F., Maniatis, T. (1989). Molecular Cloning. A Laboratory Manual. 2
nd
ed. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, New York. 18.47-18.61.