The instantaneous monitoring of polyacrylamide gels during electrophoresis.
The instantaneous monitoring of polyacrylamide gels during electrophoresis.
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电泳过程中聚丙烯酰胺凝胶的瞬时监测。
DOI：
10.1042/bj1590743
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发表时间：
1976
影响因子：
4.1
通讯作者：
A. Elliott
中科院分区：
文献类型：
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作者：
A. Elliott
指出了在电泳过程中(因此在染色前)能够看到凝胶中的蛋白质带的优点，并描述了一种依赖于这些带中的局部折射率增量的方法。简要讨论了利用聚丙烯酰胺凝胶中折射率的局部增量来测量电泳区的蛋白质浓度；发现当十二烷基硫酸钠存在时，这样的增量比蛋白质的量大得多。增强效果取决于运行条件和时间。这使得定量估计变得困难，但由于十二烷基硫酸钠的这一性质，通过基于折射率变化的观测来检测蛋白质带的灵敏度大大提高。描述了制备光学均匀凝胶(具有均匀和分级浓度的聚丙烯酰胺)的方法，这是观察折射率微小变化所必需的。描述了一种简单的暗场观测系统。给出的例子显示了在电泳过程中用该系统观察到的蛋白质样品，并与运行完成后考马斯亮蓝染色的相同凝胶进行了比较。在最佳条件下，光学方法的灵敏度与染色相当。与较低摩尔质量的蛋白质。)15000)，光学方法不是那么灵敏，随着运行时间的延长而变得不那么灵敏。使用浓度更高的聚丙烯酰胺凝胶可以大大降低灵敏度的损失，因此梯度凝胶比均匀凝胶更适合于光学观察。在电泳过程中观察蛋白质带不会增加制作染色凝胶所需的时间，而且在从染色凝胶中获得蛋白质带之前很久就能提供很多信息。
The advantages of being able to see protein zones in a gel during electrophoresis (and hence before staining) are pointed out, and a method is described which depends on local increments of refractive index in these zones. The use of local increments of refractive index in polyacrylamide gels for measuring protein concentrations in zones during electrophoresis is briefly considered; it is found that such increments are greater than would be expected from the amount of protein when sodium dodecyl sulphate is present. The enhancement depends on conditions and time of running. This makes quantitative estimates difficult, but the sensitivity of detection of protein zones by observations based on refractive-index changes is greatly increased by this property of sodium dodecyl sulphate. Methods are described for making optically uniform gels (both with uniform and with graded concentrations of polyacrylamide), necessary for observation of small changes in refractive index. A simple dark-field system of observation is described. Examples are given showing protein samples observed with the system during electrophoresis and compared with the same gel stained with Coomassie Blue after completion of the run. Under optimal conditions the optical method is comparable in sensitivity with staining. With the proteins of lower mol.wt. (approx. 15000), the optical method is not so sensitive, becoming less sensitive with longer running time. This loss of sensitivity is greatly decreased by using more concentrated polyacrylamide gels, and graded gels are therefore more suitable for optical observation than are uniform gels. The observation of protein zones during electrophoresis adds nothing to the time needed for making a stained gel and gives much information long before it can be obtained from the stained gel.