Determination of the labeling density of fluorophore-biomolecule conjugates with absorption spectroscopy

Bioconjug Chem. 2012 Feb 15;23(2):287-92. doi: 10.1021/bc2003428. Epub 2012 Jan 20.

Abstract

Dye-biomolecule conjugation is frequently accompanied by considerable spectral changes of the dye's absorption spectrum that limit the use of the common photometrical method for the determination of labeling densities. Here, we describe an improvement of this method using the integral absorbance of the dye instead of its absorbance at the long wavelength maximum to determine the concentration of the biomolecule-coupled dye. This approach is illustrated for three different cyanine dyes conjugated to the antibody IgG.

MeSH terms

  • Carbocyanines / chemistry*
  • Immunoglobulin G / chemistry*
  • Molecular Structure
  • Spectrum Analysis
  • Staining and Labeling*

Substances

  • BEBO cyanine dye
  • Carbocyanines
  • Immunoglobulin G