Research Article

Tracing pathway activities with kinase inhibitors and reverse phase protein arrays

  1. Jan van Oostrum Professor1,*,
  2. Claudio Calonder1,
  3. Daniel Rechsteiner1,
  4. Markus Ehrat1,
  5. Jürgen Mestan2,
  6. Doriano Fabbro2,
  7. Hans Voshol3

Article first published online: 9 DEC 2008

DOI: 10.1002/prca.200800070

Issue

PROTEOMICS - Clinical Applications

PROTEOMICS - Clinical Applications

Special Issue: Focus on Analysis of Drug Effects and Actions

Volume 3, Issue 4, pages 412–422, No. 4 April 2009

Additional Information

How to Cite

van Oostrum, J., Calonder, C., Rechsteiner, D., Ehrat, M., Mestan, J., Fabbro, D. and Voshol, H. (2009), Tracing pathway activities with kinase inhibitors and reverse phase protein arrays. PROTEOMICS - Clinical Applications, 3: 412–422. doi: 10.1002/prca.200800070

Author Information

  1. 1

    Zeptosens – a Division of Bayer (Schweiz) AG, Witterswil, Switzerland

  2. 2

    Expertise Platform Kinases, Novartis Institutes for BioMedical Research, Novartis AG, Basel, Switzerland

  3. 3

    Developmental and Molecular Pathways, Novartis Institutes for BioMedical Research, Novartis AG, Basel, Switzerland

*Zeptosens – a Division of Bayer (Schweiz) AG, Benkenstrasse 254, CH-4108 Witterswil, Switzerland Fax: +41-61-726-81-70

Publication History

  1. Issue published online: 30 MAR 2009
  2. Article first published online: 9 DEC 2008

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Keywords:

  • Antibodies;
  • Kinase inhibitors;
  • Phosphoproteomics;
  • Protein arrays;
  • Signaling pathways

Abstract

Controlling aberrant protein kinase activity is a promising strategy for a variety of diseases, particularly cancer. Hence, the development of kinase inhibitors is currently a focal point for pharmaceutical research. In this study we utilize a chip-based reverse phase protein array (RPA) platform for profiling of kinase inhibitors in cell-based assays. In combination with the planar wave-guide technology the assay system has an absolute LOD down to the low zeptomole range. A431 cell lysates were analyzed for the activation state of key effectors in the epidermal growth factor (EGF) and insulin signaling pathways to validate this model for compound screening. A microtiter-plate format for growing, treating, and lysing cells was shown to be suitable for this approach, establishing the value of the technology as a screening tool for characterization of large numbers of kinase inhibitors against a wide variety of cellular signaling pathways. Moreover, the reverse array format allows rapid development of site-specific phosphorylation assays, since in contrast to ELISA type systems only a single antigen-specific antibody is required.

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