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Stable Isotope Labelling with Amino Acids in Cell Culture for Human Embryonic Stem Cell Proteomic Analysis

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Human Embryonic Stem Cells Handbook

Part of the book series: Methods in Molecular Biology ((MIMB,volume 873))

Abstract

The identification and quantitative measurements of proteins in human embryonic stem cells (hESC) is a fast growing interdisciplinary area with an enormous impact on understanding the biology of hESC and the mechanism controlling self-renewal and differentiation. Using a quantitative mass spectroscopic method of stable isotope labelling with amino acids during cell culture (SILAC), we are able to analyse differential expression of proteins from different cellular compartments and to identify intracellular signalling pathways involved in self-renewal and differentiation. In this chapter, we provide a detailed method for creating SILAC media suitable for use in hESC experiments, additionally we describe methods for the isolation of membrane fractions and cytosolic and nuclear/membrane fractions.

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Author information

Authors and Affiliations

  1. Molecular Endocrinology Laboratory (KMEB), Odense University Hospital, Odense, Denmark

    Linda Harkness & Moustapha Kassem

  2. Center for Experimental Bioinformatics, University of Southern Denmark, Odense, Denmark

    Tatyana A. Prokhorova

  3. Centre for Experimental Bioinformatics, University of Southern Denmark, Odense, Denmark

    Blagoy Blagoev

Authors
  1. Linda Harkness
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  2. Tatyana A. Prokhorova
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  3. Moustapha Kassem
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  4. Blagoy Blagoev
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Corresponding author

Correspondence to Linda Harkness .

Editor information

Editors and Affiliations

  1. Sprott Centre for Stem Cell Research, Regenerative Medicine Program, Ottawa Hospital Research Institute, Smyth Road 501, Ottawa, K1Y 8L6, Canada

    Kursad Turksen

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Harkness, L., Prokhorova, T.A., Kassem, M., Blagoev, B. (2012). Stable Isotope Labelling with Amino Acids in Cell Culture for Human Embryonic Stem Cell Proteomic Analysis. In: Turksen, K. (eds) Human Embryonic Stem Cells Handbook. Methods in Molecular Biology, vol 873. Humana Press, Totowa, NJ. https://doi.org/10.1007/978-1-61779-794-1_20

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  • DOI: https://doi.org/10.1007/978-1-61779-794-1_20

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  • Publisher Name: Humana Press, Totowa, NJ

  • Print ISBN: 978-1-61779-793-4

  • Online ISBN: 978-1-61779-794-1

  • eBook Packages: Springer Protocols

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