A new approach to isoelectric focusing and fractionation of proteins in a pH gradient.

This paper describes a new method of condensation (focusing) of extended volumes of mixtures of proteins (or other ampholytes) into an isoelectric spectrum of discrete zones located at points of a pH gradient corresponding to the pI value of the individual proteins. In contradistinction to the currently practiced isoelectric focusing in "natural" pH gradients which may require as much as 96 hours for completion, the present method yields clear-cut condensations marking the isoelectric pH within about five minutes and complete fractionation within about 15 minutes. No "Ampholines" or other special buffers are required and establishment of the pH gradient requires only about 10 seconds rather than days as is common in natural pH gradient focusing. The pH gradient is generated by utilization of the temperature dependence of pH. By establishing a temperature gradient between 0 degrees and 50 degrees C in an electrophoretic column, a stable pH gradient extending over 1 pH unit can be maintained in the absence as well as the presence of a current. The pH gradient can be easily controlled by variation of the temperature limits so that high resolving power can be achieved in shallow pH gradients. The pI of the focused fractions is determined by measurement (by a thermistor or resistance thermometer) of the local temperature within each of the isoelectric zones that determine the local pH. The individual zones can be pipetted out. The method is illustrated by simultaneous condensation and evacuation of hemoglobin in two pH gradients traversed by opposite currents and by separation of hemoglobins A and S in 16 minutes in a pH gradient where the current passes in the direction of increasing pH in Tris buffer solutions stabilized by a sucrose density gradient.