Requirements for DOL measurement
Measure absorbance after removing free dye. Record dilution and path length, and correct the dye contribution at the protein quantification wavelength. DOL is the average dye count per antibody; it does not describe labeling sites or molecule-to-molecule uniformity.
Select conditions by assay performance
Titrate candidate conjugates and compare positive and appropriate negative samples under fixed instrument settings. Favor conditions that separate specific signal from background while retaining binding. Record recovery alongside these results rather than selecting by high DOL alone.
Molar-input calculation example
10 mg of IgG at 160,000 g/mol contains 62.5 nmol. A 15–25 µL aliquot of 10 mM dye contains 150–250 nmol, giving a 2.4–4:1 input ratio. This is a molar-conversion example, not a universal recommended input range.
Record input ratio separately from DOL
The input ratio is the moles of dye added per mole of target. DOL is the average number of dyes attached per target molecule after purification. Unreacted, hydrolyzed or removed dye does not contribute to conjugate DOL. A suggested IgG DOL is not an input ratio.
Calculate DOL from absorbance
Subtract the buffer blank and remove free dye first. These equations require A280 and Adye from the same diluted sample and path length. Measure Adye at the dye quantification wavelength. Use dye-specific CF280 and εdye and target-specific εprotein. Corrected A280 must be positive.
| Quantity | Equation and meaning |
|---|---|
| Corrected protein absorbance | A280,corr = A280 − CF280 × Adye |
| Protein concentration | Cprotein = A280,corr × D ÷ (εprotein × l) |
| Dye concentration | Cdye = Adye × D ÷ (εdye × l) |
| Average labeling density | DOL = Cdye ÷ Cprotein |
| Units | D: dilution factor; l: path length (cm); ε: M⁻¹ cm⁻¹; C: mol/L |