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Fast, continuous separation of mitochondria from rat myoblasts using micro free-flow electrophoresis (muFFE) with online laser-induced fluorescence detection (LIF) is reported. Mitochondrial electrophoretic profiles were acquired in less than 30 s. In comparison to macroscale FFE instruments, muFFE devices consumed approximately 100-fold less sample, used 10-fold less buffer, and required a 15-fold lower electric field. Mitochondrial electrophoretic mobility distributions measured using muFFE were compared to those measured with a capillary electrophoresis instrument with laser-induced fluorescence detection (CE-LIF). There was high similarity between the two distributions with CE-LIF distribution being offset by 1.8 x 10(-4) cm(2) V(-1) s(-1) with respect to the microFFE distribution. We hypothesize that this offset results from the differences in electric field strength used in the techniques. In comparison to CE-LIF, analysis of mitochondria using muFFE greatly decreased separation time and required less separation voltage, while maintaining low sample (125 nL) and buffer (250 microL) volumes. These features together with the potential for collecting separated organelle fractions for further characterization make microFFE a very attractive tool for the high-throughput analysis of organelle subpopulations as well as investigating the fundamentals of the electrophoretic mobility of biological particles.

Fast Determination of Mitochondria Electrophoretic Mobility Using Micro Free-Flow Electrophoresis