Improved protein separation by microchip isoelectric focusing with stepwise gradient of electric field strength
Improved protein separation by microchip isoelectric focusing with stepwise gradient of electric field strength
复制标题
通过电场强度逐步梯度微芯片等电聚焦改进蛋白质分离
DOI：
10.1002/jssc.200800514
复制
发表时间：
2009-02-01
影响因子：
3.1
通讯作者：
Zhang, Yukui
中科院分区：
文献类型：
--
作者：
Cong, Yongzheng;Liang, Yu;Zhang, Yukui
提出了基于微芯片等电聚焦的蛋白质分离采用电场强度逐步梯度的方法，在低电压聚焦后，逐步施加较高的分离电压进行等电聚焦。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的电压呈负但非线性相关。基于上述结果，在永久涂覆有聚丙烯酰胺的直玻璃微芯片通道中，应用160至1500 V/cm的逐步梯度电场强度来分离从大肠杆菌中提取的蛋白质。与在750 V/cm恒定场强下进行的传统分离相比，逐步增加的电场强度梯度提高了分辨率并减少了聚焦时间，同时没有焦耳热对蛋白质分离的负面影响。所有这些结果表明，这种方法对于实现微芯片等电聚焦复杂蛋白质样品的高分辨率和高通量分析具有重要意义。
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.