Determination of the labeling density of fluorophore-biomolecule conjugates with absorption spectroscopy.
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.
BAM Federal Institute for Materials Research and Testing, Division Biophotonic, Richard-Willstaetter-Str. 11, D-12489 Berlin, Germany.
Markus
M
Grabolle
Robert
R
Brehm
Jutta
J
Pauli
Franziska M
FM
Dees
Ingrid
I
Hilger
Ute
U
Resch-Genger
D002232:Carbocyanines / Q000737:chemistry*; D007074:Immunoglobulin G / Q000737:chemistry*; D015394:Molecular Structure; D013057:Spectrum Analysis; D013194:Staining and Labeling
C478828:BEBO cyanine dye; D002232:Carbocyanines; D007074:Immunoglobulin G