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

Authors
  • Ys, Sun
  • Jp, Landry
  • Yy, Fei
  • Xd, Zhu
  • Jt, Luo
  • Xb, Wang
  • Kit S. Lam
Type
Published Article
Journal
Langmuir
Publisher
American Chemical Society
Volume
24
Issue
23
Identifiers
DOI: 10.1021/la802097z
Source
Kit Lam Lab
License
Unknown

Abstract

We studied the effect of fluorescently labeling proteins on protein-ligand reactions. Unlabeled ligands (streptavidin-binding peptides and rabbit immunoglobulin G (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 oblique-incidence 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 K(D) = 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.

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