Receptor Tyrosine Kinase Inhibitor Profiling Using Bead-Based Multiplex Sandwich Immunoassays

  • Oliver PötzEmail author
  • Nicole Schneiderhan-Marra
  • Tanja Henzler
  • Thomas Herget
  • Thomas O. Joos
Part of the Methods in Molecular Biology book series (MIMB, volume 795)


Receptor tyrosine kinases (RTK) are important targets in drug discovery processes. Studying the phosphorylation pattern of RTKs enables the determination of their activation and inactivation states. Multiplex bead-based sandwich immunoassays are powerful tools for measuring the phosphorylation state of key regulators within cellular signalling networks. Here, we describe the analysis of the phosphorylation state of receptor tyrosine kinases using the epidermal growth factor receptor (EGFR) as an example. We provide a protocol for a bead-based sandwich immunoassay that enables a relative quantification of the EGFR and its generic tyrosine phosphorylation. We also present data from a kinase inhibitor experiment using 96-well cell-culture plates and a commercially available kit for the analysis of seven receptor tyrosine kinases.

Key words

Receptor tyrosine kinases Multiplex bead-based sandwich immunoassay Antibody array 



We would like to thank Thomas Schreiber and Jutta Bachmann for proofreading this manuscript.


  1. 1.
    Gschwind, A., Fischer, O. M., and Ullrich, A. (2004) The discovery of receptor tyrosine kinases: targets for cancer therapy, Nat Rev Cancer 4, 361–370.PubMedCrossRefGoogle Scholar
  2. 2.
    Schlessinger, J. (2000) Cell signaling by receptor tyrosine kinases. Cell 103, 211–225.PubMedCrossRefGoogle Scholar
  3. 3.
    Venter, D. J., Tuzi, N. L., Kumar, S., and Gullick, W. J. (1987) Overexpression of the c-erbB-2 oncoprotein in human breast carcinomas: immunohistological assessment correlates with gene amplification. Lancet 2, 69–72.PubMedCrossRefGoogle Scholar
  4. 4.
    Carson, R. T., and Vignali, D. A. (1999) Simultaneous quantitation of 15 cytokines using a multiplexed flow cytometric assay. J. Immunol. Methods 227, 41–52.PubMedCrossRefGoogle Scholar
  5. 5.
    Fulton, R. J., McDade, R. L., Smith, P. L., Kienker, L. J., and Kettman, J. R., Jr. (1997) Advanced multiplexed analysis with the FlowMetrix system. Clin. Chem. 43, 1749–1756.PubMedGoogle Scholar
  6. 6.
    Du, J., Bernasconi, P., Clauser, K. R., Mani, D. R., Finn, S. P., Beroukhim, R., Burns, M., Julian, B., Peng, X. P., Hieronymus, H., Maglathlin, R. L., Lewis, T. A., Liau, L. M., Nghiemphu, P., Mellinghoff, I. K., Louis, D. N., Loda, M., Carr, S. A., Kung, A. L., and Golub, T. R. (2009) Bead-based profiling of tyrosine kinase phosphorylation identifies SRC as a potential target for glioblastoma therapy. Nat. Biotechnol. 27, 77–83.PubMedCrossRefGoogle Scholar
  7. 7.
    Poetz, O., Henzler, T., Hartmann, M., Kazmaier, C., Templin, M. F., Herget, T., and Joos, T. O. (2010) Sequential multiplex analyte capturing for phosphoprotein profiling. Mol. Cell Proteomics 9, 2474–2481.PubMedCrossRefGoogle Scholar
  8. 8.
    Fry, D. W., Bridges, A. J., Denny, W. A., Doherty, A., Greis, K. D., Hicks, J. L., Hook, K. E., Keller, P. R., Leopold, W. R., Loo, J. A., McNamara, D. J., Nelson, J. M., Sherwood, V., Smaill, J. B., Trumpp-Kallmeyer, S., and Dobrusin, E. M. (1998) Specific, irreversible inactivation of the epidermal growth factor receptor and erbB2, by a new class of tyrosine kinase inhibitor. Proc. Natl. Acad. Sci. USA 95, 12022–12027.PubMedCrossRefGoogle Scholar

Copyright information

© Springer Science+Business Media, LLC 2012

Authors and Affiliations

  • Oliver Pötz
    • 1
    Email author
  • Nicole Schneiderhan-Marra
    • 1
  • Tanja Henzler
    • 2
  • Thomas Herget
    • 2
  • Thomas O. Joos
    • 1
  1. 1.NMI Natural and Medical Sciences Institute at the University of TuebingenReutlingenGermany
  2. 2.Merck KGaADarmstadtGermany

Personalised recommendations