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Microchip free flow isoelectric focusing for protein prefractionation using monolith with immobilized pH gradient
Author(s) -
Han Bin,
Wang Pingli,
Zhu Guijie,
Zhang Lihua,
Qu Feng,
Deng Yulin,
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.200800572
Subject(s) - monolith , immobilized ph gradient , chromatography , isoelectric focusing , chemistry , monolithic hplc column , free flow electrophoresis , electrophoresis , analytical chemistry (journal) , high performance liquid chromatography , polyacrylamide gel electrophoresis , biochemistry , enzyme , catalysis , gel electrophoresis of proteins
Microchip free flow IEF (μFFIEF) with monolithic IPG was proposed for protein prefractionation. The monolithic materials were first prepared by UV irradiation in a microchamber of 43 mm length, 23 mm width, and 38 μm depth. Carrier ampholytes (CAs) were further immobilized on the monolith by chemical bonding, to form a stable pH gradient. By such a technique, the continuous introduction of CAs in traditional μFFIEF could be avoided, not only to decrease the operation cost, but also to avoid the interference of CAs for the further protein identification by MS/MS. With a fluorescence microscope as the detector, under the optimal conditions, the separation of FITC labeled β‐lactoglobulin and carbonic anhydrase, with 0.9 unit difference on p I , was achieved with good reproducibility. The experimental results demonstrated that under the experimental conditions we applied, the separation mechanism of μFFIEF with M‐IPG materials might be the cooperative effects of IEF and CZE, and the former one plays a predominant role.