title: Improved protein separation by microchip isoelectric focusing with stepwise gradient of electric field strength.
abstract: 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.
authors.affiliation: 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.
authors.forename: Yongzheng
authors.initials: Y
authors.lastname: Cong
authors.forename: Yu
authors.initials: Y
authors.lastname: Liang
authors.forename: Lihua
authors.initials: L
authors.lastname: Zhang
authors.forename: Weibing
authors.initials: W
authors.lastname: Zhang
authors.forename: Yukui
authors.initials: Y
authors.lastname: Zhang
mesh_terms: D004583:Electrons; D046209:Electrophoresis, Microchip / Q000379:methods*; D007525:Isoelectric Focusing / Q000379:methods*; D011506:Proteins / Q000737:chemistry* / Q000302:isolation & purification*
chemical_list: D011506:Proteins