C.B.S. Scientific EBX-700 User Manual
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SECTION 4
Applications & Running Conditions for EBX-700 Electro-Blotting
4.1 Recommended Buffers, Power Settings and Transfer Times
1. Using 2 freezer blocks and chilled buffer, start run at 60Volts(c) @ 370mA for 1-3 hours. Run
time will vary depending on thickness of gel and sample size.
2. Buffers: ClearPAGE Transfer Buffer (cat. # CB82500) made up as follows:
COMPONENT
AMOUNT NEEDED FOR EBX-700
ClearPAGE Transfer Buffer
100 ml
Methanol
200 ml
Ultrapure water
720 ml
If you prefer to make up your own transfer buffer please make up 1X Towbin Buffer plus
0.1% SDS:
Protein Electro-Blotting Buffer:
TG-SDS (1X) 20% MeOH:
0.025M Tris base
0.192M Glycine
0.05-0.1% (w/v) SDS
20% (v/v) Methanol
4.2
References
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.
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.
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.
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.
Tesfaigzi, J., Smith-Harrison, W., and Carlson, D.M. (1994). A simple method for reusing western blots
on PVDF membranes. Biotechniques. 17:268-269.
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.
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.