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Proteostasis pp 161–175Cite as

Isolation of the Ubiquitin-Proteome from Tumor Cell Lines and Primary Cells Using TUBEs

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Part of the book series: Methods in Molecular Biology ((MIMB,volume 1449))

Abstract

Tandem ubiquitin-binding entities (TUBEs) act as molecular traps to isolate polyubiquitylated proteins facilitating the study of this highly reversible posttranslational modification. We provide here sample preparation and adaptations required for TUBE-based enrichment of the ubiquitin proteome from tumor cell lines or primary cells. Our protocol is suitable to identify ubiquitin substrates, enzymes involved in the ubiquitin proteasome pathway, as well as proteasome subunits by mass spectrometry. This protocol was adapted to prepare affinity columns, reduce background, and improve the protein recovery depending on the sample source and necessities.

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Acknowledgments

We thank Valérie Lang for the critical reading of this document, Gaël Roué for providing MCL cell lines, and Mikel Azkargorta and Felix Elortza for their technical support. This work was funded by the Ministerio de Economia y Competitividad, Spain grant BFU2011-28536 (MSR), Diputación Foral de Gipuzkoa (MSR and FA), and GSK OPEN-LAB foundation. LMC was supported by the GSK OPEN-LAB foundation. The authors would like to acknowledge networking support by the Proteostasis COST Action (BM1307).

Author information

Authors and Affiliations

  1. Proteomics Platform CICbioGUNE, CIBERehd, ProteoRed-ISCIII, Parque Tecnologico de Bizkaia, Derio, Spain

    Wendy Xolalpa

  2. Ubiquitylation and Cancer Molecular Biology, Inbiomed, San Sebastian, Spain

    Lydia Mata-Cantero & Fabienne Aillet

  3. IPBS, Université de Toulouse, CNRS, UPS and ITAV, Université de Toulouse, CNRS, UPS, 1 Place Pierre Potier, Oncopole entrée B, Toulouse, 31106, France

    Manuel S. Rodriguez

Authors
  1. Wendy Xolalpa
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  2. Lydia Mata-Cantero
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  3. Fabienne Aillet
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  4. Manuel S. Rodriguez
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Corresponding author

Correspondence to Manuel S. Rodriguez .

Editor information

Editors and Affiliations

  1. Computational and Experimental Biology Group Department of Health Promotion and Chronic Diseases, National Health Institute Dr. Ricardo Jorge, INSA, I.P., Lisboa, Portugal

    Rune Matthiesen

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© 2016 Springer Science+Business Media New York

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Xolalpa, W., Mata-Cantero, L., Aillet, F., Rodriguez, M.S. (2016). Isolation of the Ubiquitin-Proteome from Tumor Cell Lines and Primary Cells Using TUBEs. In: Matthiesen, R. (eds) Proteostasis. Methods in Molecular Biology, vol 1449. Humana Press, New York, NY. https://doi.org/10.1007/978-1-4939-3756-1_8

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  • DOI: https://doi.org/10.1007/978-1-4939-3756-1_8

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  • Publisher Name: Humana Press, New York, NY

  • Print ISBN: 978-1-4939-3754-7

  • Online ISBN: 978-1-4939-3756-1

  • eBook Packages: Springer Protocols

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