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等电聚焦电泳(IEF)是一种在一个pH梯度内操作的特殊电泳。IEF是表征蛋白质电荷不均一性的强有力工具。在一个pH梯度内,酸碱两性物(例如蛋白质)在电场的作用下会被分离,并最后在pH梯度内pH值与两性物等电点(pI)值相等的地方被聚焦成很窄的区段。IEF的分辨率在所有基于电荷的分离技术中是最高的。薄层凝胶IEF从它在上世纪70年代初被发明的时间起就成为在生物实验室被广泛应用的分析技术。但薄层凝胶IEF的主要问题是不能做定量分析,全靠手工操作及分析速度低。当毛细管IEF(cIEF)在1985年被报道时,科学家们就都立即认为cIEF能够克服IEF所有的问题。虽然cIEF看起来有很多优势,但是20多年后的今天它并没有被生物实验室广泛接受为薄层凝胶IEF的替代技术。究其原因是使用通用毛细电泳仪器的cIEF在操作上有很多困难。这种技术在IEF过程后还需要一个额外的移动过程来把聚在毛细管分离柱内的蛋白质区段推到检测器的检测点。这是由于这些毛细电泳仪器都是使用单点检测器。检测点都是靠近毛细管的一端。这个移动过程破坏IEF的高分辨率,降低重现性并使得分析速度变慢,造成在方法的优化上耗很多时间。全柱检测cIEF是在1992年提出的。该技术结合了凝胶IEF及毛细管分离的全部优点;如定量和自动化。它的分离速度大大提高。本文简单综述cIEF对不同生物实验室样品的应用。这些样品包括单克隆抗体,重组蛋白质,蛋白质复合物及失活病毒。最后,本文简述cIEF理论模拟计算的新进展。
Isoelectric focusing (IEF) is a special electrophoresis operating within a pH gradient. IEF is a powerful tool for characterizing the heterogeneity of protein charges. Within a pH gradient, acid-base amphiphiles (eg, proteins) are separated by the action of an electric field and are finally focused narrowly to a point where the pH equals the isoelectric point (pI) of the amphiphile within the pH gradient Section. IEF’s resolution is the highest among all charge-based separation technologies. The thin-layer gel IEF has been the analytical technology widely used in biological laboratories since it was first invented in the early 1970s. However, the main problem with the thin gel IEF is that it can not be done quantitatively, and it can be done manually and with low analysis speed. When the capillary IEF (cIEF) was reported in 1985, scientists immediately assumed that cIEF could overcome all IEF problems. Although cIEF appears to have many advantages, it has not been widely accepted by biological laboratories more than 20 years ago as an alternative to thin-gel IEF. The reason for this is that cIEFs using universal capillary electrophoresis instruments have many operational difficulties. This technique also requires an additional moving process after the IEF process to push the protein segments that are trapped inside the capillary column to the detector’s detection point. This is because these capillary electrophoresis instruments use single-point detectors. The test point is near the end of the capillary. This moving process destroys the high resolution of the IEF, degrading reproducibility and slowing the analysis, resulting in a lot of time spent optimizing the method. Full column detection cIEF was proposed in 1992. This technology combines the full benefits of gel IEF and capillary separation; such as quantitation and automation. Its separation speed is greatly improved. This article gives a brief overview of cIEF applications to different biological laboratory samples. These samples include monoclonal antibodies, recombinant proteins, protein complexes and inactivated viruses. Finally, this article briefly describes the recent advances in cIEF theoretical simulation.