Protein separation in pH gradients using free‐flow electrophoretic apparatus II. The pH gradients formed by the concentration gradient of boric acid in solutions of borax and mannitol

Abstract

AbstractProtein separation was investigated in the free‐flow apparatus using pH gradients generated by a concentration gradient of boric acid in a solution of borax. The linear pH gradient covering the range pH 8.2–9.2 was obtained in a solution of 2.5 mM borax due to the formation of the logarithmic concentration gradient of 0–45 mM boric acid. These pH gradients lacked a conductivity gradient in contrast to borate mannitol pH gradients, described in the joint report. By addition of mannitol into the gradient, at constant concentration, the pH was shifted toward more acidic values in proportion to the amount of mannitol added, without changing the range and shape of the pH gradient. According to this procedure of gradient formation the pH gradients can be obtained within the range from pH 3.5 to 9.2. The gradients were rapidly decaying under the experimental conditions. Thus, isoelectric focusing of proteins could not be achieved, and the separation resulted from electrophoresis in the pH gradient. The results of protein separation in the gradients practically did not depend on the point of injection of the samples into the pH gradient, at least in the cases when the proteins were injected near their isoelectric points.