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Protein fractionation in a multicompartment device using Off‐Gel™ isoelectric focusing
Author(s) -
Michel Philippe E.,
Reymond Frédéric,
Arnaud Isabelle L.,
Josserand Jacques,
Girault Hubert H.,
Rossier Joel S.
Publication year - 2003
Publication title -
electrophoresis
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 0.666
H-Index - 158
eISSN - 1522-2683
pISSN - 0173-0835
DOI - 10.1002/elps.200390030
Subject(s) - isoelectric point , isoelectric focusing , fractionation , chromatography , chemistry , immobilized ph gradient , electrophoresis , protein purification , two dimensional gel electrophoresis , resolution (logic) , mass spectrometry , proteomics , biochemistry , computer science , enzyme , artificial intelligence , gene
A new protein fractionation technique based on off‐gel isoelectric focusing (IEF) is presented, where the proteins are separated according to their isoelectric point (p I ) in a multiwell device with the advantage to be directly recovered in solution for further analysis. The protein fractions obtained with this technique have then been characterized with polymer nanoelectrospray for mass spectrometry (MS) analyses or with Bioanalyzer for mass identification. This methodology shows the possibility of developing alternatives to the classical two‐dimensional (2‐D) gel electrophoresis. One species numerical simulation of the electric field distribution during off‐gel separation is also presented in order to demonstrate the principle of the purification. Experiments with p I protein markers have been carried out in order to highlight the kinetics and the efficiency of the technique. Moreover, the resolution of the fractionation was shown to be 0.1 pH unit for the separation of β‐lactoglobulin A and B. In addition, the isoelectric fractionation of an Escherichia coli extract was performed in standard solubilization buffer to demonstrate the performances of the technique, notably for proteomics applications.