Protein separation in pH gradients using free‐flow electrophoretic apparatus I. The borate‐mannitol pH gradients

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Abstract
Protein separation in borate‐mannitol pH gradients using a free‐flow electrophoretic apparatus was studied. The pH gradients covering 0.6–1 pH unit were created by dilution of mannitol solution in borax with distilled water. Linear concentration gradients of mannitol and borax, an almost linear pH gradient, and a linear conductivity gradient, increasing from the alkaline toward the acidic end of the pH gradient, were obtained. By varying the initial mannitol concentration in borax, the gradients can be formed within the pH range 4.5–9.2. Under the experimental conditions the gradients were rapidly changed due to the influence of the electric field. The character of changes was dependent on the properties of the membranes between the electrode compartments and separation chamber, on residence time, voltage and presence of salts in the gradients. Because of gradient instability, isoelectric focusing was not achieved under the experimental conditions, and the protein separation resulted from electrophoresis in the pH gradient. The results of the separation were dependent on the point of injection of the sample into the pH gradient: the best separation was achieved when the proteins were injected above their isoelectric points.