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

Langmuir. 2008 Dec 2;24(23):13399-405. doi: 10.1021/la802097z.


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.

Publication types

  • Research Support, N.I.H., Extramural

MeSH terms

  • Animals
  • Carbocyanines / chemistry*
  • Epoxy Compounds / chemistry
  • Glass / chemistry
  • Immunoglobulin G / chemistry*
  • Kinetics
  • Ligands
  • Oligopeptides / chemistry*
  • Rabbits
  • Streptavidin / chemistry*
  • Surface Properties
  • Time Factors


  • Carbocyanines
  • Epoxy Compounds
  • Immunoglobulin G
  • Ligands
  • Oligopeptides
  • cyanine dye 3
  • Streptavidin