A novel gel electrophoretic extraction and fractionation technique in comparative analysis of functional differences and alterations in cellular metabolism.
A novel gel electrophoretic extraction and fractionation technique is described. The technique enables extraction of aqueous sample components (nonamphoteric and amphoteric) into polar and water-miscible organic solvents. The three major fractions obtained are acidic, basic, and neutral/nonextractable. In the acidic and basic fractions, the extractable compounds form a pH gradient in which they are further separated by the difference in their dissociation constants. Selective separation of acidic and basic ampholytes from other sample components is possible by modifying experimental conditions. The workup technique is particularly suitable for sample profiling because of quantitative extraction yields. Applications of the technique are shown by gas chromatographic profiles of extracellular fluids from cultured and biopsied intact cells. Differences in metabolic consumption and excretion in various cell lines and in normal and diseased cells (biopsies) as reflected in the extracellular environment are demonstrated. Profiles of extracellular fluids bathing approximately 1500 cells indicate the potential of this approach and methodology.