Protein 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 ofgradient 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 ofthe samples into the pH gradient, at least in the cases when the proteins were injected near their isoelectric points.
Protein concentration using step pH and conductivity gradients in a free-flow electrophoretic apparatus is described. The experiments were carried out in a vertical planar 36.5 x 5.0 x 0.05 cm separation chamber with 48 channels both at the inlet and outlet. The gradients were generated by injecting a 2.5 mM borax solution (pH 9.2, conductivity = 0.5 mS) into the chamber via channels No. 1-12 counting from the cathode, and 0.45 M boric acid (pH 4.3, conductivity = 0.025 mS) together with the protein to be concentrated via the remaining channels. The gradients were suitable for the concentration of the proteins with isoelectric points 2 pH 7.0 (human immunoglobulin G, bovine hemoglobin). From mixtures of hemoglobin and albumin, the latter was removed during concentration. The protein concentration of the starting solution does not affect the results of concentration. At constant current of 30 mA and a residence time of 45 s, the proteins were concentrated at a rate of 400 ml of starting solution per hour with a maximum concentration factor of 10.
I IntroductionElectrophoretic methods can be used not only for fractionation but also for concentration ofproteins. Procedures and apparatus were developed for protein extraction from gel slices with simultaneous concentration, following the separation in polyacrylamide gel. Electrophoresis in combination with isoeiectric focusing [ 11 can extract proteins from gel slices with additional purification and concentration in the pH gradient. Moving boundary electrophoresis 121 for protein extraction and concentration offers a number of advantages, and buffer systems suitable for different proteins have been described [31. For largevolumes, obtained e. g. during protein purification, concentration by isoelectric focusing under the conditions excluding protein inactivation on membranes has been applied [4-71. Concentration has been achieved in density gradient columns with a fairly drastic pH step, either innonlinear pH gradients generated with amino acids and synthetic carrier ampholytes [41 or in step pH gradients formed with a borax-boric acid-polyhydroxylic compound buffer system [5-71. The initially uniformly distributed proteins were concentrated from a definite column volume at their isoelectric points in the region of the drastic pH step. Both a concentration as well as a removal of contaminating protein with other isoelectric points could be achieved [4-71. Concentration in density gradient columns is afflicted with some inconveniences. (i) The protein concentration of the starting solution must be low, otherwise the proteins may precipitate at their isoelectric points and sediment in the column, disturbing the concentration process. (ii) In a single experiment only limited volumes of the starting solution can be concentrated depending on the dimensions of the columns. (iii) The duration ofthe experiment is usually several hours although in most applications it would be desirable to achieve a concentration in a shorter time.Application of free-flow apparatus could over...
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