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Bacterial Protein Secretion Systems pp 321–351Cite as

Blue Native PAGE Analysis of Bacterial Secretion Complexes

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Part of the book series: Methods in Molecular Biology ((MIMB,volume 1615))

Abstract

Bacterial protein secretion systems serve to translocate substrate proteins across up to three biological membranes, a task accomplished by hydrophobic, membrane-spanning macromolecular complexes. The overexpression, purification, and biochemical characterization of these complexes is often difficult, impeding progress in understanding the structure and function of these systems. Blue native (BN) polyacrylamide gel electrophoresis (PAGE) allows for the investigation of these transmembrane complexes right from their originating membranes, without the need for long preparative steps, and is amenable to the parallel characterization of a number of samples under near-native conditions. Here we present protocols for sample preparation, one-dimensional BN PAGE and two-dimensional BN/sodium dodecyl sulfate (SDS)-PAGE, as well as for downstream analysis by staining, immunoblotting, and mass spectrometry on the example of the type III secretion system encoded on Salmonella pathogenicity island 1.

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Acknowledgments

Work performed in the laboratory of SW was supported by the Alexander von Humboldt Foundation in the framework of the Sofja Kovalevskaja Award endowed by the Federal Ministry of Education and Research (BMBF) and in the framework of the Georg Forster Research Fellowships (to J.V.M.F.), and by the Deutsche Forschungsgemeinschaft (DFG) as part of the Collaborative Research Center (SFB) 766 Bacterial cell envelope, project B14.

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Authors and Affiliations

  1. Section of Cellular and Molecular Microbiology, Interfaculty Institute of Microbiology and Infection Medicine (IMIT), University of Tübingen, Elfriede-Aulhorn-Str. 6, 72076, Tübingen, Germany

    Susann Zilkenat, Tobias Dietsche, Julia V. Monjarás Feria, Claudia E. Torres-Vargas, Mehari Tesfazgi Mebrhatu & Samuel Wagner

  2. German Center for Infection Research (DZIF), Partner-site Tübingen, Elfriede-Aulhorn-Str. 6, 72076, Tübingen, Germany

    Samuel Wagner

Authors
  1. Susann Zilkenat
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  2. Tobias Dietsche
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  3. Julia V. Monjarás Feria
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  4. Claudia E. Torres-Vargas
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  5. Mehari Tesfazgi Mebrhatu
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  6. Samuel Wagner
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Corresponding author

Correspondence to Samuel Wagner .

Editor information

Editors and Affiliations

  1. Institut de Microbiologie de la Méditerranée, Aix-Marseille Univ - CNRS, Marseille, France

    Laure Journet

  2. Institut de Microbiologie de la Méditerranée, Aix-Marseille Univ - CNRS, Marseille, France

    Eric Cascales

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Zilkenat, S., Dietsche, T., Monjarás Feria, J.V., Torres-Vargas, C.E., Mebrhatu, M.T., Wagner, S. (2017). Blue Native PAGE Analysis of Bacterial Secretion Complexes. In: Journet, L., Cascales, E. (eds) Bacterial Protein Secretion Systems. Methods in Molecular Biology, vol 1615. Humana Press, New York, NY. https://doi.org/10.1007/978-1-4939-7033-9_26

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  • DOI: https://doi.org/10.1007/978-1-4939-7033-9_26

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  • Publisher Name: Humana Press, New York, NY

  • Print ISBN: 978-1-4939-7031-5

  • Online ISBN: 978-1-4939-7033-9

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