One- and Two-Dimensional Miniaturized Electrophoresis of Proteins with Native Fluorescence Detection

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Abstract
Miniaturized electrophoresis was successfully coupled with native fluorescence detection for direct analysis of proteins in one- and two-dimensional separations. The detection setup was based on direct observation of the UV-induced fluorescence of proteins using a CCD camera and a Hg (Xe) lamp for sample excitation. Protein mixtures were readily separated by size on a 1-cm segment of the one-dimensional gel in 8 min, and a detection limit of 0.04 ng per band was achieved. The dynamic range of the system was larger than 2 orders of magnitude. Miniaturized slab gel electrophoresis was performed on a special holder designed to couple isoelectric focusing with SDS−PAGE. Two-dimensional separation, including rehydration of IEF strip and fluorescence detection was completed in 2.5 h. Approximately 200 protein spots from Escherichia coli were detected on a 1 cm2 area. A detection limit of 0.1 μg of total protein was achieved. The operation should be amenable to total automation.