Cong Yongzheng, Liang Yu, Zhang Lihua, Zhang Weibing, Zhang Yukui
National Chromatographic R. and A. Center, Key Lab of Separation Science for Analytical Chemistry, Dalian Institute of Chemical Physics, Chinese Academy of Sciences, Dalian, China.
J Sep Sci. 2009 Feb;32(3):462-5. doi: 10.1002/jssc.200800514.
A stepwise gradient of electric field strength was proposed for microchip IEF-based protein separation, by which after focusing at low voltages, IEF was performed by applying higher separation voltages step-by-step. A linear relationship between the focusing time and the inverse of the electric field strength was found. In addition, the conductivity of an established pH gradient showed a negative but nonlinear correlation with the applied voltage. Based on the above-mentioned results, a stepwise gradient of electric field strength, ranging from 160 to 1500 V/cm was applied in the separation of proteins extracted from Escherichia coli in a straight glass microchip channel permanently coated by polyacrylamide. Compared to the conventional separation performed under a constant field strength of 750 V/cm, the increased stepwise gradient of electric field strength resulted in improved resolution and decreased focusing time, while without the negative effects of Joule heat for protein separation. All these results demonstrated that such a method might be of great significance to achieve high resolution and high-throughput analysis of complex protein samples for microchip IEF.
有人提出了一种用于基于微芯片等电聚焦的蛋白质分离的电场强度逐步梯度法，通过该方法，在低电压下聚焦后，逐步施加更高的分离电压来进行等电聚焦。发现聚焦时间与电场强度的倒数之间存在线性关系。此外，已建立的pH梯度的电导率与施加电压呈负相关但非线性相关。基于上述结果，在由聚丙烯酰胺永久涂层的直玻璃微芯片通道中，对从大肠杆菌中提取的蛋白质进行分离时，施加了范围为160至1500 V/cm的电场强度逐步梯度。与在750 V/cm的恒定场强下进行的传统分离相比，电场强度逐步梯度的增加导致分辨率提高和聚焦时间缩短，同时对蛋白质分离没有焦耳热的负面影响。所有这些结果表明，这种方法对于实现微芯片等电聚焦对复杂蛋白质样品的高分辨率和高通量分析可能具有重要意义。
