THEORETICAL AND EXPERIMENTAL-STUDY OF HIGH-PERFORMANCE ELECTROPHORETIC MOBILIZATION OF ISOELECTRICALLY FOCUSED PROTEIN ZONES
THEORETICAL AND EXPERIMENTAL-STUDY OF HIGH-PERFORMANCE ELECTROPHORETIC MOBILIZATION OF ISOELECTRICALLY FOCUSED PROTEIN ZONES
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DOI：
10.1016/s0021-9673(01)94519-4
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发表时间：
1987-01-30
期刊：
影响因子：
--
通讯作者：
YAO, K
中科院分区：
文献类型：
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作者：
HJERTEN, S;LIAO, JL;YAO, K
在早期的一篇论文中，我们证明了有可能动员一系列等电聚焦的蛋白质，从而在试管内或试管外检测它们。动员是以不同的方式进行的，例如通过将阳极液交换为阴极液或将阴极液交换为阴极液进行电泳法。本文从电中性条件出发，从理论上处理营养动员问题。这样获得的信息被用来设计适当组成的阳极体和阴极体，用于动员聚焦蛋白。通过在长度为110 mm的玻璃管中进行的自由溶液聚焦-动员实验，说明了这些电极溶液的有用性。由于管子的内径和壁厚只有0.05 mm，焦耳热量被有效地去除，从而允许使用高场强(270V/cm)。因此，聚焦时间短至6分钟。动员时间约为15min(360V/cm)。通过280 nm处的吸光度测量，在试管上检测到被动员的蛋白质带。用非交联型聚丙烯酰胺对玻璃管进行处理，以消除电渗透和蛋白质在管壁上的吸附。通过实验验证了理论研究得出的以下结论：通过选择含有质子(羟基离子)以外的阳离子(阴离子)的阳极液(阴极液)，可以实现向阳(阴)极的动员；然后阳离子(阴离子)将以电泳法迁移到分离管中，并从管的阳(阴)端持续增加(降低)pH。发生动员的电极溶液的pH值对于管外检测是关键的，但对于管上检测则不是。借助于等电聚焦中的电中性条件，可以很容易地解释所谓平台现象的产生。
In an earlier paper we showed that it is possible to mobilize a train of isoelectrically focused proteins and thus detect them on-tube or off-tube. The mobilization was performed in different ways, for instance electrophoretically by exchanging the anolyte for the catholyte or vice versa. In this paper we treat the elctrophoretic mobilization theoretically, originating from the conditions of electroneutrality. The information thus gained was used to design anolytes and catholytes of appropriate compositions for mobilization of focused proteins. The usefulness of these electrode solutions is illustrated by focusing-mobilization experiments performed in free solution in a glass tube of length 110 mm. Since the inside diameter of the tube and its wall thickness were only 0.05 mm, the Joule heat was efficiently removed, which allowed the use of high field strengths (270 V/cm. The focusing time was therefore as short as 6 min. The time required for mobilization was about 15 min (360 V/cm). The mobilized protein zones were detected on-tube by absorbance measurements at 280 nm. The glass tube was treated with non-cross-linked polyacrylamide to eliminate electroendosmosis and adsorption of proteins onto the tube wall. The following conclusions drawn from the theoretical studies were experimentally verified: mobilization toward the anode (cathode) can be accomplished by selecting an anolyte (catholyte) containing a cation (anion) other than the proton (hydroxyl ion); the cation (anion) will then electrophoretically migrate into the separation tube and continuously increase (decrease) the pH from the anodic (cathodic) end of the tube. The pH of the electrode solution toward which the mobilization takes place is critical for off-tube, but not for on-tube detection. With the aid of the electroneutrality condition that applies in isoelectric focusing, one can easily explain the generation of the so-called plateau phenomenon.