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Fast determination of mitochondria electrophoretic mobility using micro free-flow electrophoresis.

ABSTRACT: 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.

SUBMITTER: Kostal V

PROVIDER: S-EPMC2779522 | biostudies-literature | 2009 Nov

REPOSITORIES: biostudies-literature

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Fast determination of mitochondria electrophoretic mobility using micro free-flow electrophoresis.

Kostal Vratislav V Fonslow Bryan R BR Arriaga Edgar A EA Bowser Michael T MT

Analytical chemistry 20091101 22

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 we ...[more]

PMID: 19908903

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Project description:BackgroundHypocrea jecorina (anamorph Trichoderma reesei) is a filamentous ascomycete of industrial importance due to its hydrolases (e.g., xylanases and cellulases). The regulation of gene expression can influence the composition of the hydrolase cocktail, and thus, transcription factors are a major target of current research. Here, we design an approach for identifying a repressor of a xylanase-encoding gene.ResultsWe used streptavidin affinity chromatography to isolate the Xylanase promoter-binding protein 1 (Xpp1). The optimal conditions and templates for the chromatography step were chosen according to the results of an electrophoretic mobility shift assay performed under repressing conditions, which yielded a DNA-protein complex specific to the AGAA-box (the previously identified, tetranucleotide cis-acting element). After isolating AGAA-box binding proteins, the eluted proteins were identified with Nano-HPLC/tandem MS-coupled detection. We compared the identified peptides to sequences in the H. jecorina genome and predicted in silico the function and DNA-binding ability of the identified proteins. With the results from these analyses, we eliminated all but three candidate proteins. We verified the transcription of these candidates and tested their ability to specifically bind the AGAA-box. In the end, only one candidate protein remained. We generated this protein with in vitro translation and used an EMSA to demonstrate the existence of an AGAA-box-specific protein-DNA complex. We found that the expression of this gene is elevated under repressing conditions relative to de-repressing or inducing conditions.ConclusionsWe identified a putative transcription factor that is potentially involved in repressing xylanase 2 expression. We also identified two additional potential regulatory proteins that bind to the xyn2 promoter. Thus, we succeeded in identifying novel, putative transcription factors for the regulation of xylanase expression in H. jecorina.

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Fast determination of mitochondria electrophoretic mobility using micro free-flow electrophoresis.

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Fast determination of mitochondria electrophoretic mobility using micro free-flow electrophoresis.

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