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A bead-based western for high-throughput cellular signal transduction analyses

Author

Listed:
  • Fridolin Treindl

    (NMI Natural and Medical Sciences Institute at the University of Tübingen
    Pharmaceutical Biotechnology, Eberhard-Karls-Universität Tübingen)

  • Benjamin Ruprecht

    (Chair for Proteomics and Bioanalytics, Center of Life and Food Sciences Weihenstephan, Technische Universität München
    Center for Integrated Protein Science Munich)

  • Yvonne Beiter

    (NMI Natural and Medical Sciences Institute at the University of Tübingen
    Pharmaceutical Biotechnology, Eberhard-Karls-Universität Tübingen)

  • Silke Schultz

    (Medical Faculty and University Hospital of the Heinrich-Heine University Duesseldorf)

  • Anette Döttinger

    (NMI Natural and Medical Sciences Institute at the University of Tübingen)

  • Annette Staebler

    (University Hospital Tübingen)

  • Thomas O. Joos

    (NMI Natural and Medical Sciences Institute at the University of Tübingen)

  • Simon Kling

    (NMI Natural and Medical Sciences Institute at the University of Tübingen)

  • Oliver Poetz

    (NMI Natural and Medical Sciences Institute at the University of Tübingen)

  • Tanja Fehm

    (Medical Faculty and University Hospital of the Heinrich-Heine University Duesseldorf)

  • Hans Neubauer

    (Medical Faculty and University Hospital of the Heinrich-Heine University Duesseldorf)

  • Bernhard Kuster

    (Chair for Proteomics and Bioanalytics, Center of Life and Food Sciences Weihenstephan, Technische Universität München
    Center for Integrated Protein Science Munich
    Bavarian Biomolecular Mass Spectrometry Center (BayBioMS), Technische Universität München)

  • Markus F. Templin

    (NMI Natural and Medical Sciences Institute at the University of Tübingen
    Pharmaceutical Biotechnology, Eberhard-Karls-Universität Tübingen)

Abstract

Dissecting cellular signalling requires the analysis of large number of proteins. The DigiWest approach we describe here transfers the western blot to a bead-based microarray platform. By combining gel-based protein separation with immobilization on microspheres, hundreds of replicas of the initial blot are created, thus enabling the comprehensive analysis of limited material, such as cells collected by laser capture microdissection, and extending traditional western blotting to reach proteomic scales. The combination of molecular weight resolution, sensitivity and signal linearity on an automated platform enables the rapid quantification of hundreds of specific proteins and protein modifications in complex samples. This high-throughput western blot approach allowed us to identify and characterize alterations in cellular signal transduction that occur during the development of resistance to the kinase inhibitor Lapatinib, revealing major changes in the activation state of Ephrin-mediated signalling and a central role for p53-controlled processes.

Suggested Citation

  • Fridolin Treindl & Benjamin Ruprecht & Yvonne Beiter & Silke Schultz & Anette Döttinger & Annette Staebler & Thomas O. Joos & Simon Kling & Oliver Poetz & Tanja Fehm & Hans Neubauer & Bernhard Kuster , 2016. "A bead-based western for high-throughput cellular signal transduction analyses," Nature Communications, Nature, vol. 7(1), pages 1-11, November.
    • Handle: RePEc:nat:natcom:v:7:y:2016:i:1:d:10.1038_ncomms12852
    DOI: 10.1038/ncomms12852
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    Cited by:

    1. Stephan Spahn & Fabian Kleinhenz & Ekaterina Shevchenko & Aaron Stahl & Yvonne Rasen & Christine Geisler & Kristina Ruhm & Marion Klaumuenzer & Thales Kronenberger & Stefan A. Laufer & Holly Sundberg-, 2024. "The molecular interaction pattern of lenvatinib enables inhibition of wild-type or kinase-mutated FGFR2-driven cholangiocarcinoma," Nature Communications, Nature, vol. 15(1), pages 1-13, December.

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