Capillary isoelectric focusing with pH 9.7 cathode for the analysis of gastric biopsies

Abstract

Capillary isoelectric focusing tends to suffer from poor reproducibility, particularly for the analysis of complex protein samples from cellular or tissue homogenates. This poor reproducibility appears to be associated with erratic variations 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. Another cause of variation is protein adsorption to the capillary wall, which causes an increase in electroosmotic flow. The effects of protein adsorption can be reduced by use of surfactants in the buffer and by employing an extremely low sample loading. 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.

Fig 1

Capillary isoelectric focusing separation of proteins from a biopsy sample, labeled with Chromeo P540. Sodium hydroxide was employed at the cathode end. The sheath-flow consisted of phosphoric acid (2m mM, pH 3). Proteins were mobilized with NH₄OH. All experiments were performed on an AAP coated capillary. A. Runs 1, 3, and 6. B. Close-up of Runs 1 and 6. C. Current profiles for the three runs.

Fig 2

Capillary isoelectric focusing separation of proteins from a biopsy sample, labeled with Chromeo P540. Sodium hydroxide was replaced with CAPS at the cathode end. The sheath-flow consisted of phosphoric acid (2m mM, pH 3). Proteins were mobilized with NH₄OH. All experiments were performed on an AAP coated capillary. A. Five runs taken over a three-day period. B. Close-up of Runs 10 and 20. C. Current profiles for the runs.