Single cell analysis by capillary electrophoresis

Understanding the molecular mechanisms of many fundamental cell biology processes requires analysis of chemical species within single cell. Development and application of micro-scale analytical techniques such as capillary electrophoresis with laser induced fluorescence detection, and crude cell extract and single cell handling and labeling approaches were presented in this thesis in an effort to address a few of the challenging goals of single cell research.; A single intact cell is injected into the separation capillary. Cellular and organelle membranes are lysed releasing the cellular contents into the separation buffer. High voltage is applied to the injection end of the capillary to separate the contents by electrophoresis.; A simple proteome map was obtained from a HT29 colon adenocarcinoma cell. The protein content was fluorescently labeled with the fluorogenic reagent 3-(2-furoyl)quinoline-2-carboxaldehyde. Several dozen components were resolved. A number of experiments were done to verify that these components were proteins. Protein expression varied significantly between cells, but the average expression was consistent with that observed from a protein extract prepared from 106 cells. This technique is also correlated cell cycle with oligosaccharide metabolic activity in single cells by measuring DNA ploidy, the uptake of a fluorescent disaccharide, and the amount of metabolic products in a single cell.; Two new approaches to concentrate proteins during the labeling process were discussed in this thesis. The first method combines the concentration of stacking, reaction efficiency of on-capillary labeling, and the separation efficiency of CZE. The response for selected protein fractions was enhanced through manipulation of sample stacking conditions. The second method presents isotachophoretic stacking of proteins, which is induced by using excess labeling reagent as the leading electrolyte.