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J Biochem Biophys Methods. 1999 Feb 25;39(1-2):93-110.

Capillary isoelectric focusing with whole column imaging detection for analysis of proteins and peptides.

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Department of Chemistry, University of Waterloo, Ontario, Canada.


Whole column imaging detection has been developed for capillary isoelectric focusing (CIEF) of proteins and peptides. In this imaged C1EF technique, a solution of sample and ampholytes was introduced into a short (4-5 cm), internally coated capillary stabilized by a cartridge. After applying high DC voltage, the isoelectric focusing process takes place and the focused zones are monitored in a real-time mode using the imaging detectors developed. Three types of imaging detectors have been developed including refractive index gradient, laser-induced fluorescence (LIF), and absorption. Of these, absorption imaging detection is the most practical at the present time due to its quantitative ability and universal characteristics. Whole column imaging detection eliminates the mobilization step required for single point detection after the focusing process. Therefore, it provides a fast analysis speed (3-5 min for each sample), and avoids the disadvantages associated with the mobilization process, such as distortion of pH gradient and loss in resolution. In this paper, we review the methodology of imaged CIEF as well as progress in instrumental development, IEF performed on a microchip, and the application to protein and peptide analysis.

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