Theoretical and experimental study of high-performance electrophoretic mobilization of isoelectrically focused protein zones.

In an earlier paper we showed that it is possible to mobilize a train of isoelectrically focused proteins and thus detect them on-tube or off-tube. The mobilization was performed in different ways, for instance electrophoretically by exchanging the anolyte for the catholyte or vice versa. In this paper we treat the electrophoretic mobilization theoretically, originating from the conditions of electroneutrality. The information thus gained was used to design anolytes and catholytes of appropriate compositions for mobilization of focused proteins. The usefulness of these electrode solutions is illustrated by focusing-mobilization experiments performed in free solution in a glass tube of length 110 mm. Since the inside diameter of the tube and its wall thickness were only 0.05 mm, the Joule heat was efficiently removed, which allowed the use of high field strengths (270 V/cm). The focusing time was therefore as short as 6 min. The time required for mobilization was about 15 min (360 V/cm). The mobilized protein zones were detected on-tube by absorbance measurements at 280 nm. The glass tube was treated with non-cross-linked polyacrylamide to eliminate electroendosmosis and adsorption of proteins onto the tube wall. The following conclusions drawn from the theoretical studies were experimentally verified: mobilization toward the anode (cathode) can be accomplished by selecting an anolyte (catholyte) containing a cation (anion) other than the proton (hydroxyl ion); the cation (anion) will then electrophoretically migrate into the separation tube and continuously increase (decrease) the pH from the anodic (cathodic) end of the tube. The pH of the electrode solution toward which the mobilization takes place is critical for off-tube, but not for on-tube detection. With the aid of the electroneutrality condition that applies in isoelectric focusing, one can easily explain the generation of the so-called plateau phenomenon.