Original Article

Determination of binding curves via protein micropatterning in vitro and in living cells

  1. Stefan Sunzenauer1,
  2. Verena Zojer3,
  3. Mario Brameshuber2,
  4. Andreas Tröls2,
  5. Julian Weghuber4,
  6. Hannes Stockinger3,
  7. Gerhard J. Schütz1,2,*

Article first published online: 2 NOV 2012

DOI: 10.1002/cyto.a.22225

Additional Information

How to Cite

Sunzenauer, S., Zojer, V., Brameshuber, M., Tröls, A., Weghuber, J., Stockinger, H. and Schütz, G. J. (2012), Determination of binding curves via protein micropatterning in vitro and in living cells. Cytometry. doi: 10.1002/cyto.a.22225

Author Information

  1. 1

    Biophysics Institute, Johannes Kepler University Linz, A-4040 Linz, Austria

  2. 2

    Institute of Applied Physics, Vienna University of Technology, 1040 Vienna, Austria

  3. 3

    Molecular Immunology Unit, Institute for Hygiene and Applied Immunology, Center for Pathophysiology, Infectiology, and Immunology, Medical University of Vienna, A-1090 Vienna, Austria

  4. 4

    School of Engineering and Environmental Sciences, University of Applied Sciences Upper Austria, 4600 Wels, Austria

*Institute of Applied Physics, Vienna University of Technology, Wiedner Hauptstrasse 8-10, 1040 Vienna, Austria

Publication History

  1. Article first published online: 2 NOV 2012
  2. Manuscript Accepted: 9 OCT 2012
  3. Manuscript Revised: 23 AUG 2012
  4. Manuscript Received: 30 MAY 2012

Funded by

  • GEN-AU Project of the Austrian Federal Ministry for Science and Research
  • Austrian Science Fund. Grant Number: Projects Y250-B03 and I301-B12
  • DOC-fellowship of the Austrian Academy of Sciences at the Institute of Biophysics

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Keywords:

  • Micropatterning;
  • plasma membrane;
  • CD4;
  • Lck;
  • single molecule microscopy;
  • equilibrium binding constant

Abstract

Quantification of protein interactions in living cells is of key relevance for understanding cellular signaling. With current techniques, however, it is difficult to determine binding affinities and stoichiometries of protein complexes in the plasma membrane. We introduce here protein micropatterning as a convenient and versatile method for such investigations. Cells are grown on surfaces containing micropatterns of capture antibody to a bait protein, so that the bait gets rearranged in the live cell plasma membrane. Upon interaction with the bait, the fluorescent prey follows the micropatterns, which can be readout with fluorescence microscopy. In this study, we addressed the interaction between Lck and CD4, two central proteins in early T-cell signaling. Binding curves were recorded using the natural fluctuations in the Lck expression levels. Surprisingly, the binding was not saturable up to the highest Lck expression levels: on average, a single CD4 molecule recruited more than nine Lck molecules. We discuss the data in view of protein- and lipid-mediated interactions. © 2012 International Society for Advancement of Cytometry

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