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Stable Isotope Labeling by Amino Acids in Cell Culture (SILAC) pp 23–46Cite as

SILAC Labeling of Yeast for the Study of Membrane Protein Complexes

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

Abstract

Despite their simplicity compared to multicellular organisms, single-celled yeasts such as the baker’s yeast Saccharomyces cerevisiae are widely recognized as model organisms for the study of eukaryotic cell biology. To gain deeper insights into the molecular mechanisms underlying cellular processes, it is of utmost interest to establish the interactome of distinct proteins and to thoroughly analyze the composition of individual protein complexes and their dynamics. Combining affinity purification of epitope-tagged proteins with high-resolution mass spectrometry and quantitative proteomics strategies, in particular stable isotope labeling by amino acids in cell culture (SILAC), represents an unbiased and powerful approach for a most accurate characterization of protein complexes. In this chapter, we provide detailed protocols for the generation of yeast strains (S. cerevisiae) amenable to SILAC-labeling, for epitope tagging of a protein of interest for affinity purification, and for the SILAC-based characterization of membrane protein complexes including the identification of stable core components and transient interaction partners.

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Acknowledgments

This work was supported by the Deutsche Forschungsgemein-schaft, Forschergruppe 1905 and the Excellence Initiative of the German Federal and State Governments (EXC 294 BIOSS).

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Authors and Affiliations

  1. Department of Biochemistry and Functional Proteomics, Faculty of Biology, and BIOSS Centre for Biological Signalling Studies, University of Freiburg, Schänzlestr. 1, Freiburg, 79104, Germany

    Silke Oeljeklaus, Andreas Schummer, Ida Suppanz & Bettina Warscheid

Authors
  1. Silke Oeljeklaus
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  2. Andreas Schummer
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  3. Ida Suppanz
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  4. Bettina Warscheid
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Corresponding author

Correspondence to Bettina Warscheid .

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Editors and Affiliations

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

    Bettina Warscheid

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Oeljeklaus, S., Schummer, A., Suppanz, I., Warscheid, B. (2014). SILAC Labeling of Yeast for the Study of Membrane Protein Complexes. 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_3

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  • DOI: https://doi.org/10.1007/978-1-4939-1142-4_3

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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

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