Amino Acids, Peptides and Proteins - Dévényi T., Gergely J. 1976

Medium- and High-Voltage Electrophoresis Methods
High-Voltage Electrophoresis Method

In high-voltage Electrophoresis, the voltage gradient is 50—100 V/cm. The current applied also increases, consequently leading to greater heat generation and a rise in the Temperature of the paper. More efficient cooling is required to dissipate the generated heat.

In one type of horizontal high-voltage electrophoresis apparatus, cooling is achieved by circulating tap Water through a metal cooling plate. Naturally, the metal plate must be insulated from the filter paper by a polyethylene film with appropriate insulating properties. In water-cooled high-voltage electrophoresis systems, the filter paper is cooled from both above and below by two plates pressed together by an inflatable rubber ring. If stitching was used during the experiment, the paper must be covered with a layer of foam plastic to ensure uniform pressure, but this compromises the cooling effect of one of the plates. The portions of the paper sheet extending beyond the cooling plate receive no cooling at all. Therefore, for instance, the filter paper wick immersed in the electrode compartment must be double-layered to rapidly compensate for the loss of buffer solution due to heating and prevent the paper from drying out.

Another group of high-voltage electrophoresis apparatuses is designed so that the filter paper is immersed in a water-immiscible coolant; the heat absorbed by this liquid is then dissipated by a water-cooling system. In these devices, the paper is placed vertically between two buffer reservoirs. Due to this vertical arrangement, selecting the correct electrode polarity is crucial. Electroendosmosis causes the buffer solution to migrate from the anode to the cathode. If, for example, the negative pole of the power supply is connected to the upper reservoir, the specific 'tailing' during electrophoresis caused by the upward electroendosmotic flow will be fully compensated by the non-specific 'tailing' resulting from the downward gravity-driven movement of the buffer solution.

A good coolant must be immiscible with water, have a high heat capacity, and a high flash point. Drying of the paper during electrophoresis increases electrical resistance in the circuit, which in turn can lead to spark discharge. If the coolant has a low flash point, a fire hazard arises, as the electrophoresis chamber is filled with 50—80 l of flammable liquid. Toluene is commonly used as a coolant; it has suitable thermal conductivity but a very low flash point and is therefore highly flammable. Unlike toluene, the petroleum fraction commercially known as 'white spirit' has a fairly high flash point. Carbon tetrachloride can also be used; it is non-flammable but has poor thermal conductivity.

A significant advantage of high-voltage electrophoresis apparatuses is that they substantially reduce the duration of the run, thereby minimizing diffusion and yielding sharper zone boundaries.

All Methods described for medium-voltage electrophoresis can be reproduced on high-voltage electrophoresis apparatuses without any modifications. The only exception is the dual-buffer system. As is well known, in direct liquid-cooled systems, the buffer solution in the reservoirs saturates the coolant, and an equilibrium is established between the buffer solution and the paper during electrophoresis. Consequently, stabilizing two buffer systems on a single sheet of paper becomes impossible.

In direct liquid-cooled electrophoresis apparatuses, the same reservoir must be used for a given buffer solution; changing the buffer solution once a week is sufficient. A buffer solution with a different pH can only be used with a fresh batch of coolant.

References

1. Brown J. R., Hartley B. S., Biochem. J., 101, 214 (1966).

2. Butler R. J. G., Harris J. I., Hartley B. S., Leberman R., Biochem. J., 103, 78P (1967).

3. Devenyi T., Magy. Kem. Folyóirat, 69, 538 (1963).

4. Dixon H. B. F., Perham R. N., Biochem. J., 109, 312 (1968).

5. Goldberger R. F., Anfinsen C. B., Biochemistry, 1, 401 (1962).

6. Ingram V. M., Nature, 180, 326 (1957).

7. Mühlrad A., Hegyi Gy., Toth G., Acta Biochim. Biophys. Acad. Sci. Hung., 2, 19 (1967).

8. Naughton M., Hagopian H., Anal. Biochem., 3, 276 (1962).

9. Ovádi J., Libor S., Elődi P., Acta Biochim. Biophys. Acad. Sci. Hung., 2, 455 (1967).

10. Perham R. N., Jones G. M. T., Eur. J. Biochem., 2, 87 (1967).

11. Tang J., Hartley B. S., Biochem. J., 102, 593 (1967).

Recommended Literature

Bailey L. J., Techniques in Protein Chemistry, Elsevier Publ. Co., Amsterdam, London, New York, 1967.

Hartley R. J., Biochem. J., 119, 805 (1970).

Leach S. J., ed., Physical Principles and Techniques of Protein Chemistry, Part A, Academic Press, New York, London, 1969.

Smith I., Chromatographic and Electrophoretic Techniques II, Zone electrophoresis, Heinemann Medical Books Ltd., London, 1960.

Zweig G., Whitaker J. R., Paper Chromatography and Electrophoresis, Vol. I, Academic Press, New York, London, 1967.