Run-to-run irreproducibility is a major issue plaguing cIEF for analysis of complex protein samples. Nevertheless, the small number of publications describing the application of capillary isoelectric focusing to the analysis of complex protein samples is striking. As one example, we have published an example of the analysis of a homogenate prepared from a Barrett’s esophagus cell line, which resolved many dozen components. However, there are very few studies employing cIEF for analysis of complex protein samples prepared from a cellular or tissue homogenate. Several publications employed capillary isoelectric focusing for separation of complex peptide mixtures generated from tryptic digest of yeast or E. However, the analysis of complex samples is much less common. Hemoglobin and recombinant proteins appear to be particularly commonly analytes. The addition of salt ions at one end disrupts the pH gradient and causes the analytes to migrate past the detector.Ĭapillary isoelectric focusing has most commonly been used to analyze simple samples, usually containing a single protein or a handful of standard proteins. Chemical mobilization is arguably the most efficient, and is performed by adding additional salt ions to either the anode or cathode. There are several approaches to mobilization. End-column detection allows use of high sensitivity laser-induced fluorescence or information-rich mass spectrometry as detectors. Either of two detection methods are then employed: whole-column imaging in which a camera is used to image the focused contents of the capillary, or end-column detection, where the focused analytes are mobilized past a fixed detector near one end of the capillary. In isoelectric focusing, ampholytes establish a pH gradient in the capillary and the analytes focus to their isoelectric points. We report the use of CAPS mobilization buffer in combination with an ultrasensitive laser-induced fluorescence detector for the reproducible analysis of ~2 ng of protein from a Barrett’s esophagus biopsy.Īmong capillary electrophoresis separation methods, capillary isoelectric focusing (cIEF) provides the greatest resolving power, but has typically been the most challenging technique to perform. The effects of protein adsorption can be reduced by use of surfactants in the buffer and by employing an extremely low sample loading. Another cause of variation is protein adsorption to the capillary wall, which causes an increase in electroosmotic flow. One cause of electroosmotic flow variation is degradation of the capillary coating caused by the extremely basic solution commonly used during mobilization and focusing this degradation of the capillary coating can be reduced by employing a CAPS mobilization buffer at pH 9.

This poor reproducibility appears to be associated with erratic variations in electroosmotic flow. Capillary isoelectric focusing tends to suffer from poor reproducibility, particularly for the analysis of complex protein samples from cellular or tissue homogenates.

Isoelectric focus anode cathode