Effect of fluorescently labeling protein probes on kinetics of protein-ligand reactions

Y. S. Sun, J. P. Landry, Y. Y. Fei, X. D. Zhu, J. T. Luo, X. B. Wang, Kit Lam

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75 Scopus citations

Abstract

Westudied the effect of fluorescently labeling proteins on protein-ligand reactions. Unlabeled ligands (streptaVidinbinding peptides and rabbit immunoglobulinG(IgG) as antigen targets) are immobilized on epoxy-functionalized glass slides. Unlabeled and Cy3-labeled protein probes from the same batch (streptavidin and goat antibodies) subsequently react with the surface-immobilized targets. By monitoring in situ the surface mass density change using an obliqueincidence reflectivity difference scanning microscope (a label-free detector), we measured k on and k off for streptavidin-peptide reactions and antibody-antigen reaction. We found that (1) equilibrium dissociation constants, defined as KD ) k off/k on, for streptavidin-peptide reactions increases by a factor of 3-4 when the solution-phase streptavidin is labeled with Cy3 dye and (2) K D for reactions of solution-phase goat anti-rabbit antibodies with rabbit IgG targets also change significantly when the goat antibodies are labeled with Cy3 dye.

Original languageEnglish (US)
Pages (from-to)13399-13405
Number of pages7
JournalLangmuir
Volume24
Issue number23
DOIs
StatePublished - Dec 2 2008

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ASJC Scopus subject areas

  • Electrochemistry
  • Condensed Matter Physics
  • Surfaces and Interfaces
  • Materials Science(all)
  • Spectroscopy

Cite this

Sun, Y. S., Landry, J. P., Fei, Y. Y., Zhu, X. D., Luo, J. T., Wang, X. B., & Lam, K. (2008). Effect of fluorescently labeling protein probes on kinetics of protein-ligand reactions. Langmuir, 24(23), 13399-13405. https://doi.org/10.1021/la802097z