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SILAC for the Study of Mammalian Cell Lines and Yeast Protein Complexes

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Quantitative Methods in Proteomics

Part of the book series: Methods in Molecular Biology ((MIMB,volume 893))

Abstract

Through crucial advancements in quantitative mass spectrometry (MS), proteomics has evolved from taking mere “snapshots” of proteomes to thoroughly studying dynamic changes in entire proteomes and characterizing intricate protein–protein interaction or signaling networks. Thus, quantitative MS-based proteomics offers the unique potential to place proteins into their functional context and, moreover, to improve our understanding of the molecular processes involved in the development, survival, or pathology of cells and organisms. Among the vast variety of techniques developed for the accurate quantification of proteins via MS, stable isotope labeling by amino acids in cell culture (SILAC) arguably represents the most elegant method. In this chapter, we provide a detailed protocol for the establishment of SILAC for mammalian cell culture systems. In addition, to exemplify the high versatility of SILAC for addressing different biological questions, we describe the successful “pairing” of SILAC with conventional affinity purification (AP)-MS approaches allowing for accurately characterizing protein complexes.

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Acknowledgments

The authors would like to thank Dr. Christine David and Dr. Friedel Drepper for scientific discussion as well as Christa Reichenbach, Inga Michels, and Astrid Tschapek for technical assistance. This work was supported by Deutsche Forschungsgemeinschaft, the Excellence Initiative of the German Federal and State Governments (EXC 294 BIOSS) and FOR1352.

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Authors and Affiliations

  1. Faculty of Biology and BIOSS Centre for Biological Signalling Studies, University of Freiburg, Freiburg, Germany

    Heike Piechura, Silke Oeljeklaus & Bettina Warscheid

Authors
  1. Heike Piechura
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  2. Silke Oeljeklaus
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  3. Bettina Warscheid
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Corresponding author

Correspondence to Bettina Warscheid .

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Editors and Affiliations

  1. Fak. Medizin, Medizinische Proteom Center, Universität Bochum, Universitätsstraße 150, Bochum, 44801, Germany

    Katrin Marcus

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Piechura, H., Oeljeklaus, S., Warscheid, B. (2012). SILAC for the Study of Mammalian Cell Lines and Yeast Protein Complexes. In: Marcus, K. (eds) Quantitative Methods in Proteomics. Methods in Molecular Biology, vol 893. Humana Press, Totowa, NJ. https://doi.org/10.1007/978-1-61779-885-6_14

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  • DOI: https://doi.org/10.1007/978-1-61779-885-6_14

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  • Publisher Name: Humana Press, Totowa, NJ

  • Print ISBN: 978-1-61779-884-9

  • Online ISBN: 978-1-61779-885-6

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