Righetti P G, Gelfi C
J Biochem Biophys Methods. 1984 May;9(2):103-19. doi: 10.1016/0165-022x(84)90002-2.
A further improvement on the preparative aspects of immobilized pH gradients (IPG) (J. Biochem. Biophys. Methods (1983) 8, 135-155, 157-172) is described, based on the use of soft (highly diluted) polyacrylamide gels. While in conventional IPGs in 5%T gels an upper load limit of 40-45 mg protein/ml gel volume is found, in 2.5%T gels, containing the same amount of Immobiline, as much as 90 mg protein/ml gel can be applied, without overloading effects. This is an extraordinary amount of material to be carried by a gel phase, and renders IPG by far the leading technique in any electrophoretic fractionation. A new, two-step casting technique, based on the formation of a %T step and a pH plateau around the application trench, is described. A new method for electrophoretic protein recovery from IPG gel strips, based on embedding on low-gelling agarose (37 degrees C), is reported. The physico-chemical properties of highly diluted gels, in relation to their protein loading ability, are evaluated and discussed. It is recommended that diluted gels (e.g. 3.5%T) be used also in analytical runs, since sharper protein zones are obtained, due to the increased charge density on the polymer coil.
本文介绍了基于使用软质（高度稀释）聚丙烯酰胺凝胶对固定化pH梯度（IPG）制备方法（《生物化学与生物物理方法杂志》（1983年）8卷，135 - 155页，157 - 172页）的进一步改进。在传统的5%T凝胶IPG中，发现每毫升凝胶体积的蛋白质上样上限为40 - 45毫克，而在含有相同量固定化电解质的2.5%T凝胶中，每毫升凝胶可上样高达90毫克蛋白质，且无过载效应。这是凝胶相能够承载的大量物质，使得IPG成为任何电泳分离中迄今为止领先的技术。本文描述了一种基于在加样槽周围形成%T梯度和pH平台的新的两步灌胶技术。报道了一种基于嵌入低熔点琼脂糖（37℃）从IPG凝胶条中电泳回收蛋白质的新方法。评估并讨论了高度稀释凝胶的物理化学性质与其蛋白质上样能力的关系。建议在分析运行中也使用稀释凝胶（如3.5%T），因为由于聚合物线圈上电荷密度增加，可获得更清晰的蛋白质条带。
