RAPID ISOELECTRIC FOCUSING IN DENSITY GRADIENT COLUMNS *
RAPID ISOELECTRIC FOCUSING IN DENSITY GRADIENT COLUMNS *
复制标题
密度梯度柱中的快速等电聚焦 *
DOI：
10.1111/j.1749-6632.1973.tb47520.x
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发表时间：
1973
影响因子：
5.2
通讯作者：
H. Rilbe
中科院分区：
文献类型：
--
作者：
H. Rilbe
Isoelectric focusing in natural pH gradients and artificial density gradients is usually carried out in rather slender columns with cooled vertical walls. This has the advantage of a considerable vertical extension being available for the separation of quite a number of protein zones with a satisfactory resolution even if the zones are not particularly thin. It is also technically very simple, for micropreparative purposes, to take fractions by suction through a capillary tube because the horizontal liquid flow to such an exit tube can be limited to a few millimeters. Fractions can also be taken from the bottom of the column, that, for this purpose, has a cross-sectional area gradually decreasing to the dimension of the exit tube. The density gradient exerts a stabilizing effect against convection in favor of laminar flow so that fractions emerge in correct order with a minimum of remixing. The use of a tall column, however, also has the drawback that the available density interval, about 0.2 g/cm8, has to be spread out over a considerable distance, whereby the resulting density gradient becomes correspondingly weak. Electric tension and current, then, have to be kept at relatively low values, and the separation takes a long time, 1-2 days with apparatus in general use. Philpot,l on the contrary, has advocated very short columns, strong density gradients, extremely thin protein zones, and short separation times. Unquestionably, this principle is characterized by good economy in’ time, electric power, and material; Philpot sketched a continuous flow electrophoretic separator based on these principles. Although he classed his invention “with those aeroplanes at South Kensington which never left the ground,” his article contains so many valuable viewpoints and suggestions that it is still worthy of an often repeated study. This is even more remarkable as the paper was written only 2 years after Tiselius’ breakthrough articlez of 1937 on electrophoretic analysis by the moving boundary method. As a matter of fact, in this early work Philpot introduced zonal electrophoresis 10 years before it came into general use, and density gradient electrophoresis 15 years before Kolin3,4 and Svensson and Valmeta demonstrated that a density gradient is a good means of effecting zonal electrophoresis in free liquid columns. Kolin, in reviving the method of isoelectric focusing, preferred working with very short and strong density gradients, strong currents, and rapid separation (a few minutes). However, this method suffers severely from the technical difficulty of withdrawing protein zones from the column that are thin vertically but extending over a considerable horizontal area. Also, thin zones broaden much more rapidly, relatively, than thicker zones because zone spreading is a square root function of time counted from an inflnitely thin initial zone. Kolina described the use of a plunger that reduced the cross-sectional area by a factor of 4 and increased zone thicknesses and zone separations bv the same factor.
