Fluorescence detection for gel and capillary electrophoresis

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Hogan, Barry
Major Professor
Edward S. Yeung
Committee Member
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Several separation and detection schemes for gel and capillary electrophoresis are described. Emphasis is placed upon direct and indirect fluorescence detection systems as utilized for the determination of proteins, peptides, and intracellular analytes;First, an indirect fluorescence detection system for the separation of proteins via gel electrophoresis is described. Quantities as low as 50 nanograms of bovine serum albumin and soybean trypsin inhibitor are separated and detected visually without the need for staining of the analytes. This is very similar to levels of protein commonly separated with gel electrophoresis;A separation and detection system utilizing indirect fluorescence detection with capillary zone electrophoresis is also demonstrated. Tryptic digest peptide fragments are separated and detected at the 500 attomole level without derivatization via indirect fluorescence detection. Capillary electrophoresis provides efficient separations of 10[superscript]4-10[superscript]5 plates in total separation times of three minutes. Mass detection limits are 180 times lower than comparable CE absorbance detection figures of merit;Finally, a method for analyzing the contents of a single human erythrocyte is described. Both indirect and direct fluorescence detection are used. Indirect fluorescence detection is used to detect femtomole levels of sodium and potassium released from the cytoplasm of a single human erythrocyte lysed within the CE capillary. Direct detection is used to detect attomole quantities of the tripeptide glutathione (GSH) from within the cytoplasm of a single lysed erythrocyte. GSH was derivatized with monobromobimane (mBBr), which is thiol specific. Derivatization is carried out by allowing the bimane reagent to permeate the living cell and derivatize the analyte in vitro. No tedious microchemistry or cell handling was required;Diamide and dithiothreitol were applied to living erythrocytes to modulate the quantity of intracellular GSH present in its reduced form. Single cell analysis was able to detect a statistically significant difference in the mean GSH levels of untreated cell populations, diamide treated cell populations, and diamide and dithiothreitol treated cell populations. All relevant cellular heterogeneity data was also preserved. Diamide and dithiothreitol are commonly used to model the mechanism by which toxic intracellular metabolites decrease GSH levels, thereby increasing cellular susceptibility to intracellular damage, carcinogenesis, and mutagenesis.

Wed Jan 01 00:00:00 UTC 1992