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Mycobacteria Protocols pp 111–142Cite as

Two-Dimensional Gel Electrophoresis-Based Proteomics of Mycobacteria

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Part of the book series: Methods in Molecular Biology ((MIMB,volume 465))

Abstract

Two-dimensional gel electrophoresis (2-DE) in combination with mass spectrometry (MS) is the classic proteomics approach used to monitor the dynamics of protein abundance and posttranslational modifications in biological systems. In this chapter, we provide detailed protocols for 2-DE–based proteomics of mycobacteria. Adequate standard operating procedures for mycobacterial culture, subcellular fractionation, and selective enrichment of proteins are indispensable prerequisites for targeted proteome analyses. Therefore, we also provide approved protocols for selective and efficient extraction of cytosolic, secreted, and hydrophobic plasma membrane proteins of mycobacteria, as well as for isolation of mycobacteria from infected macrophages.

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Acknowledgments

The authors thank Dr. Peter Jungblut (MPIIB), Dr. Frank Schmidt (University of Oslo, Biotechnology Centre of Oslo, Oslo, Norway), and Dr. Achim Treumann (Royal College of Surgeons, Department of Clinical Pharmacology, Dublin, Ireland) for their fruitful cooperation, Dr. Sarah Fortune (Harvard School of Public Health, Department of Immunology and Infectious Diseases, Boston, MA) for providing us with the protocol described in Section 8.3.1.3, Dr. Stephen Reece (MPIIB) for critical reading of the manuscript, and the Bundesministerium für Bildung und Forschung (Competence Network “Neue Methoden zur Erfassung des Gesamtproteoms von Bakterien” J.M., S.H.E.K., U.E.S.; Competence network “PathoGenoMik-Plus”; J.M., S.H.E.K.), the Deutsche Forschungsgemeinschaft (DFG Priority Programme SPP1131; U.E.S.), the Royal Society Wolfson Research Merit Award, UK (U.E.S.), and the European Community (Project “Structural and Functional Genomics of M. tuberculosis”; S.H.E.K.) for financial support.

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Authors and Affiliations

  1. Department of Immunology, Max Planck Institute for Infection Biology, Charitéplatz 1, D-10117, Berlin, Germany

    Jens Mattow Ph.D

  2. Department of Immunology, Max Planck Institute for Infection Biology, Berlin, Germany

    Frank Siejak, Kristine Hagens, Julia Kreuzeder, Stefan H.E. Kaufmann & Ulrich E. Schaible Ph.D

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  1. Jens Mattow Ph.D
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  2. Frank Siejak
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  3. Kristine Hagens
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  4. Julia Kreuzeder
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  5. Stefan H.E. Kaufmann
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  6. Ulrich E. Schaible Ph.D
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Correspondence to Jens Mattow Ph.D .

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Editors and Affiliations

  1. Queen Mary's School of Med. & Dentistry, Inst. Cell & Molecular Science, Barts and the London, Newark Street 4, London, E1 2AT, United Kingdom

    Tanya Parish

  2. Queen Mary's School of Med. & Dentistry, Inst. Cell & Molecular Science, Barts and the London, Newark Street 4, London, E1 2AT, United Kingdom

    Amanda Claire Brown

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Mattow, J., Siejak, F., Hagens, K., Kreuzeder, J., Kaufmann, S.H., Schaible, U.E. (2009). Two-Dimensional Gel Electrophoresis-Based Proteomics of Mycobacteria. In: Parish, T., Brown, A. (eds) Mycobacteria Protocols. Methods in Molecular Biology, vol 465. Humana Press, Totowa, NJ. https://doi.org/10.1007/978-1-59745-207-6_8

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  • DOI: https://doi.org/10.1007/978-1-59745-207-6_8

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