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Combination of Stable Isotope Labeling by Amino Acids in Cell Culture (SILAC) and Substrate Trapping for the Detection of Transient Protein Interactions

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SILAC

Part of the book series: Methods in Molecular Biology ((MIMB,volume 2603))

Abstract

Antibody-based affinity purification is a recognized method for use in studying protein–protein interactions. There are four different classes of proteins that are typically identified with such affinity purification workflows: bait protein, proteins that specifically interact with the bait protein, proteins nonspecifically associated with the antibody, and proteins that cross-react with the antibody. Mass spectrometry can be used to differentiate these classes of proteins in affinity-purified mixtures. Here we describe the use of stable isotope labeling by amino acids in cell culture, substrate trapping, and mass spectrometry to enable the objective identification of the components of affinity-purified protein complexes.

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Acknowledgments

SNT acknowledges funding from the National Institutes of Health’s National Center for Advancing Translational Sciences, grant UL1TR002494, and start-up funds from the University of Minnesota Department of Laboratory Medicine and Pathology.

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Authors and Affiliations

  1. Department of Biochemistry, Molecular Biology and Biophysics, University of Minnesota College of Biological Sciences, Minneapolis, MN, USA

    Jolene M. Duda

  2. Department of Laboratory Medicine and Pathology, University of Minnesota School of Medicine, Minneapolis, MN, USA

    Stefani N. Thomas

Authors
  1. Jolene M. Duda
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  2. Stefani N. Thomas
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Corresponding author

Correspondence to Stefani N. Thomas .

Editor information

Editors and Affiliations

  1. Analytical Chemistry Department, Faculty of Chemical Sciences, Complutense University of Madrid, Madrid, Spain

    Jose L. Luque-Garcia

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© 2023 The Author(s), under exclusive license to Springer Science+Business Media, LLC, part of Springer Nature

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Duda, J.M., Thomas, S.N. (2023). Combination of Stable Isotope Labeling by Amino Acids in Cell Culture (SILAC) and Substrate Trapping for the Detection of Transient Protein Interactions. In: Luque-Garcia, J.L. (eds) SILAC. Methods in Molecular Biology, vol 2603. Humana, New York, NY. https://doi.org/10.1007/978-1-0716-2863-8_18

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  • DOI: https://doi.org/10.1007/978-1-0716-2863-8_18

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  • Publisher Name: Humana, New York, NY

  • Print ISBN: 978-1-0716-2862-1

  • Online ISBN: 978-1-0716-2863-8

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