The degree of labeling (DOL) is the average number of dye molecules coupled to a protein molecule (e.g. an antibody). The DOL can be determined from the absorption spectrum of the labeled antibody.
For the DOL determination, the antibody conjugate solution should be gel filtrated. Then, the UV/VIS spectrum of the solution is acquired in a window starting at ~250 nm and covering the complete absorbance band of the coupled dye (see the dye data sheet provided by Abberior). The absorption spectra must be acquired using a UV transparent quartz cuvette. Read the following values from the absorption spectrum:
the wavelength where the absorbance has its maximum (should be close to the absorbance maximum of the pure dye, compare the respective data sheet)
the absorbance at λmax
the absorbance at 280 nm
The DOL is calculated from the measured values according to the following equation:
Here, the symbols denote:
absorbance of the pure protein at 280 nm (absorption maximum of proteins)
molar extinction coefficient (in M-1cm-1) of the pure protein at 280 nm
molar extinction coefficient (in M-1cm-1) of the dye at its absorbance maximum. For Abberior dyes, εmax is specified in the respective product data sheet.
correction factor which depends on the dye's spectral properties. For Abberior dyes, C280 is specified in the dye's data sheet.
molar extinction coefficient (in M-1cm-1) of the pure protein at 280 nm. Unless specified by the vendor of the antibody it must be determined in a separate measurement.
Note that the above equation is only accurate if the extinction coefficient of the free dye εmax at the maximum absorbance is equal to the value of the dye-antibody conjugate at its maximum absorbance. In practice, the calculated DOL values may be off by ~20 %.