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Identification and Validation of Protein-Protein Interactions by Combining Co-immunoprecipitation, Antigen Competition, and Stable Isotope Labeling

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Stable Isotope Labeling by Amino Acids in Cell Culture (SILAC)

Part of the book series: Methods in Molecular Biology ((MIMB,volume 1188))

Abstract

Co-immunoprecipitation (coIP) in combination with mass spectrometry (MS) is a powerful tool to identify potential protein-protein interactions. However, unspecifically precipitated proteins usually result in large numbers of false-positive identifications. Here we describe a detailed protocol particularly useful in plant sciences that is based on 15N stable isotope labeling of cells, 14N antigen titration, and coIP/MS to distinguish true from false protein-protein interactions.

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Acknowledgements

This work was supported by grants from the Deutsche For-schungsgemeinschaft (Schr 617/5-1) and the Bundesministerium für Bildung und Forschung (Systems Biology Initiative FORSYS, project GoFORSYS), and by the Max Planck Society.

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Authors and Affiliations

  1. Molecular Biotechnology and Systems Biology, TU Kaiserslautern, Paul-Ehrlich-Str. 23, 67663, Kaiserslautern, Germany

    Frederik Sommer, Timo Mühlhaus, Dorothea Hemme, Daniel Veyel & Michael Schroda

  2. Max Planck Institute of Molecular Plant Physiology, Am Muehlenberg 1, 14476, Potsdam-Golm, Germany

    Frederik Sommer, Timo Mühlhaus, Dorothea Hemme, Daniel Veyel & Michael Schroda

Authors
  1. Frederik Sommer
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  2. Timo Mühlhaus
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  3. Dorothea Hemme
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  4. Daniel Veyel
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  5. Michael Schroda
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Corresponding author

Correspondence to Michael Schroda .

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Editors and Affiliations

  1. Institute of Biology II, University of Freiburg, Freiburg, Germany

    Bettina Warscheid

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Sommer, F., Mühlhaus, T., Hemme, D., Veyel, D., Schroda, M. (2014). Identification and Validation of Protein-Protein Interactions by Combining Co-immunoprecipitation, Antigen Competition, and Stable Isotope Labeling. In: Warscheid, B. (eds) Stable Isotope Labeling by Amino Acids in Cell Culture (SILAC). Methods in Molecular Biology, vol 1188. Humana Press, New York, NY. https://doi.org/10.1007/978-1-4939-1142-4_17

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  • DOI: https://doi.org/10.1007/978-1-4939-1142-4_17

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  • Publisher Name: Humana Press, New York, NY

  • Print ISBN: 978-1-4939-1141-7

  • Online ISBN: 978-1-4939-1142-4

  • eBook Packages: Springer Protocols

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