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


Semi-dry transfer has traditionally been a fast but inefficient method for transferring all the protein from a gel. By using a discontinuous buffer system, transfer efficiency can be greatly increased. In protein blotting, the buffer has an important effect on the elution of the proteins from a gel and the retention of the protein to the membrane.

A unique feature of semi-dry blotting is the ability to use two different buffers during transfer, known as a discontinuous buffer system. This is important because the effect of methanol and SDS are opposing to both the gel and membrane respectively. Optimization of the buffer with respect to both the gel and membrane can be accomplished using a discontinuous buffer system.


The Effects of Methanol
Methanol increases a proteins affinity for a membrane by removing the SDS from the protein. This increases the available hydrophobic sites on the protein for binding to the membrane support. It is for this reason methanol is often used in transfer buffers. While advantageous for binding of the protein to the membrane, methanol causes the pores in the gel to constrict. This makes it mechanically more difficult for the protein to exit the gel.


The Effects of SDS
SDS is used in western blotting transfer buffer to aid in the elution of proteins from the gel matrix. While it inhibits the binding of the protein to the membrane, it is often necessary to facilitate complete transfer of proteins from the gel.


Using A Discontinuous Buffer System
The opposing effects of methanol and SDS in blotting can be exploited in semi-dry transfer because the buffer reservoirs, the filter paper on both sides of the gel, are independent. In a discontinuous system, methanol should be included in the buffer on the membrane side (anode) of the blot assembly and SDS used on the gel side (cathode), taking advantage of the positive effects of each component.

A new discontinuous buffer system using a Tris-CAPS buffer provides excellent results in semi-dry blotting. This new procedure uses Tris-CAPS buffer plus 15% methanol in the filter paper on the anode and Tris-CAPS plus 0.1% SDS in the filter paper on the cathode.

Cytosol from white blood cells was separated by SDS-PAGE. The phox-47 component of the Respiratory Burst Oxidase was detected by Immun-Star chemiluminescent substrate.


For Best Results
Bio-Rads Extra Thick Blotting Paper should be used. This paper is 2.6 mm thick, 100% cotton fiber, and provides the absorbency required for semi-dry blotting.

PVDF provides superior retention of the protein. For proteins less than 20 K it is required to prevent blow-through during electrophoretic transfer. Its higher binding capacity will increase detection results. It is important to first wet the PVDF in 100% methanol, then equilibrate in bottom/anode buffer for at least 30 minutes prior to transfer. It is expedient to begin equilibration during the vertical electrophoresis run.

Buffer Formulation
Tris-CAPS (60 mM Tris base, 40 mM CAPS, pH 9.6)
5x Stock Solution
36.34 g Tris-base
44.26 g CAPS
Water to 1 liter

To prepare 100 ml working buffers from 5x stock solution:
Bottom/Anode buffer: Top/Cathode bu 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. Mozdzanowski, J. and Speicher, D. W., in Current Research in Protein Chemistry (Villafranca, J., Ed.), pp.8793, Academic Press, San Diego (1990).

8. Patterson, S. D., Anal. Biochem., 221, 115 (1994).

9. Smejkal, G. and Gallagher, S., BioTechniques, 16, 196202 (1994).

10. Svoboda, M., Meuris, S., Robyn, C. and Christophe, J., Anal. Biochem., 151, 1623 (1985).

11. Tovey, E. R. and Baldo, B. A., Electrophoresis, 8, 384387 (1987).

12. Vaessen, R. T. M. J., Kreike, J. and Groot, G. S. P., FEBS Lett., 124, 193196 (1981).


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