On the relationship between applied voltage and the duration of isoelectric focusing experiments in the development of a linear pH gradient

Abstract

The development of a linear pH gradient is necessary for the precise and consistent separation of biomolecules via isoelectric focusing techniques. Existing applications have used various voltage potentials in performing the separations, but the exact relationship between the voltage and the required time duration on the development of a linear pH gradient has not been determined. Certain applications, such as separations within the microgravity environment, can benefit from the use of low voltages, whereas other applications are not constrained by voltage limitations. In general, as the voltage increases, the time duration required for high-definition separation appears to be reduced, but the effect of these changes on the linearity of the pH gradient was not previously known. A mixture of ampholytes with a pH range of 3 to 10 was used in the investigation of this relationship, and voltage levels from 125 V to 1000 V were employed. A 6% polyacrylamide gel in a cylindrical configuration was used as the medium in all the experiments. The pH profiles of the gels were determined immediately after the conclusion of each time-duration experiment, without disturbing the integrity of the gel. This was accomplished by using a specialized iridium-oxide pH sensor that can be placed directly on the gel surface. The authors present their findings on the ability to produce linear pH gradients as a function of the voltage level applied and the time duration of the isoelectric focusing experiment.