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Two‐dimensional capillary electrophoresis: Capillary isoelectric focusing and capillary zone electrophoresis with laser‐induced fluorescence detection
Author(s) -
Dickerson Jane A.,
Ramsay Lauren M.,
Dada Oluwatosin O.,
Cermak Nathan,
Dovichi Norman J.
Publication year - 2010
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.201000151
Subject(s) - isoelectric focusing , capillary electrophoresis , capillary action , chromatography , chemistry , isoelectric point , free flow electrophoresis , laser induced fluorescence , analytical chemistry (journal) , fluorescence , electrophoresis , buffer (optical fiber) , capillary electrochromatography , polyacrylamide gel electrophoresis , materials science , gel electrophoresis of proteins , optics , biochemistry , telecommunications , physics , computer science , composite material , enzyme
CIEF and CZE are coupled with LIF detection to create an ultrasensitive 2‐D separation method for proteins. In this method, two capillaries are joined through a buffer‐filled interface. Separate power supplies control the potential at the injection end of the first capillary and at the interface; the detector is held at ground potential. Proteins are labeled with the fluorogenic reagent Chromeo P503, which preserves the isoelectric point of the labeled protein. The labeled proteins were mixed with ampholytes and injected into the first‐dimension capillary. A focusing step was performed with the injection end of the capillary at high pH and the interface at low pH. To mobilize components, the interface was filled with a high pH buffer, which was compatible with the second‐dimension separation. A fraction was transferred to the second‐dimension capillary for separation. The process of fraction transfer and second dimension separation was repeated two dozen times. The separation produced a spot capacity of 125.