Weber G, Bocek P
Dr. Weber GmbH, Kirchheim, Germany.
Electrophoresis. 1998 Jul;19(10):1649-53. doi: 10.1002/elps.1150191021.
Continuous flow electrophoresis (CFE) is a promising method for preparative fractionation of a variety of biological species, ranging from peptides and proteins to subcellular particles and cells. The high separation efficiency of FFE may be deteriorated by hydrodynamic distortion of zones due to the omnipresent parabolic laminar flow profile. We show in this paper that the detrimental hydrodynamic distortion of separated proteins zones can be reduced, with resultant enhancement of separation efficiency, by employing continuous isoelectric focusing in pH gradients as the actual working regime in an advanced instrumentation. Newly developed media for fast generation of narrow- or broad-range pH gradients under CFE conditions are described. The separation efficiency of these pH gradients is comparable to that of the gradients formed with the aid of synthetic carrier ampholytes. The new media are defined mixtures of nontoxic chemicals, and thus they are compatible with the requirements of human medicine. Experimental data are given showing that the new media offer fractionation of isoforms of proteins, that they offer resolution of proteins differing in isoelectric point (pI) by less than 0.05 pH units, and that these media inhibit proteins precipitation in experiments with human serum proteins.
连续流动电泳（CFE）是一种用于多种生物物质制备分离的有前景的方法，这些生物物质范围从肽和蛋白质到亚细胞颗粒及细胞。由于普遍存在的抛物线状层流分布，FFE的高分离效率可能会因区域的流体动力学畸变而降低。我们在本文中表明，通过在先进仪器中采用pH梯度连续等电聚焦作为实际工作模式，可以减少分离的蛋白质区域有害的流体动力学畸变，从而提高分离效率。本文还描述了在CFE条件下用于快速生成窄范围或宽范围pH梯度的新开发介质。这些pH梯度的分离效率与借助合成载体两性电解质形成的梯度相当。新介质是无毒化学品的特定混合物，因此它们符合人类医学的要求。实验数据表明，新介质可对蛋白质同工型进行分离，能够分辨等电点（pI）相差小于0.05个pH单位的蛋白质，并且这些介质在人血清蛋白实验中可抑制蛋白质沉淀。
