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Indexed by:期刊论文

Date of Publication:2011-05-15

Journal:JOURNAL OF CHROMATOGRAPHY B-ANALYTICAL TECHNOLOGIES IN THE BIOMEDICAL AND LIFE SCIENCES

Included Journals:SCIE、EI

Volume:879

Issue:17-18,SI

Page Number:1439-1443

ISSN No.:1570-0232

Key Words:In situ fluorescence derivatization; Hollow fiber membrane interface; Preconcentration; Low abundance proteins

Abstract:A novel device, composed of a syringe pump for sample loading, a hydrophilic hollow fiber membrane interface for protein concentration and small molecules removal, and a centrifugation tube for buffer exchange, was designed for protein preconcentration and in situ fluorescence derivatization. With the outlet of the interface blocked, denatured proteins were continually introduced. Restricted by the membrane with the molecular weight cutoff (MWCO) of 3000 Da, proteins were concentrated within the membrane. However, denaturant and other small molecules, which might affect the further fluorescence derivatization, were driven out of the membrane. Then, the membrane with proteins restricted inside was directly put into the fluorescence derivatization buffer. Here, the water-soluble sulfo-3H-indocyanine dye, the active N-hydroxysuccinimide of 3H-indolium,1-(5-carboxypentyl)-2[3-[1-(5-carboxypentyl)-1,3-dihydro-3,3-dimethyl-5-sulfo-2H-indol-2-ylidene]-1-propenyl]-3,3-dimethy1-5-sulfo-,monopotassium salt (sw-cy3-NHS). synthesized in our lab. was used for protein labeling. By such a method, the detection sensitivity of bovine serum albumin (BSA) was improved by nearly 200 folds, compared to that obtained by direct in-solution derivatization. Through the derivatization of a fraction of E. colt protein separated by reversed phase HPLC, proteins with low concentration were efficiently labeled, which indicated the potential merit of the developed method for the high sensitive detection of low abundance proteins. (C) 2010 Elsevier B.V. All rights reserved.