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Improved protein separation by microchip isoelectric focusing with stepwise gradient of electric field strength
Author(s) -
Cong Yongzheng,
Liang Yu,
Zhang Lihua,
Zhang Weibing,
Zhang Yukui
Publication year - 2009
Publication title -
journal of separation science
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 0.72
H-Index - 102
eISSN - 1615-9314
pISSN - 1615-9306
DOI - 10.1002/jssc.200800514
Subject(s) - electric field , isoelectric focusing , voltage , field strength , chemistry , analytical chemistry (journal) , isoelectric point , resolution (logic) , chromatography , joule heating , electric field gradient , protein purification , materials science , magnetic field , physics , composite material , computer science , biochemistry , quantum mechanics , artificial intelligence , enzyme
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.