Degree of Labeling Calculator (Dye-to-Protein Ratio)

How it works

corrected A280 = A280 − Amax × CF · protein (M) = corrected A280 × dilution ÷ ε protein · dye (M) = Amax × dilution ÷ ε dye · DOL = dye (M) ÷ protein (M) · protein (mg/mL) = protein (M) × MW

After conjugating a fluorescent dye to an antibody or other protein and removing free dye, the degree of labeling — moles of dye per mole of protein — tells you whether the reaction worked. The dye absorbs at its own maximum (494 nm for Alexa Fluor 488, 650 nm for Alexa Fluor 647) and a little at 280 nm, so its contribution is removed from the A280 with the correction factor before the protein is quantified by the Beer–Lambert law. For IgG, a DOL of roughly 2–10 is typical depending on the dye; too low gives dim staining, too high causes self-quenching and can reduce antibody affinity. Extinction coefficients and correction factors follow the manufacturer’s values (Thermo Fisher).

Worked example

An IgG labeled with Alexa Fluor 488 reads A280 = 1.2 and A494 = 0.9. Corrected A280 = 1.2 − 0.9 × 0.11 = 1.101, so protein = 1.101 ÷ 210,000 = 5.243 µM (0.786 mg/mL). Dye = 0.9 ÷ 71,000 = 12.676 µM, giving a degree of labeling of 12.676 ÷ 5.243 = 2.42.

Sources: Thermo Fisher (Pierce) Tech Tip #31 — Calculate dye:protein (F/P) molar ratios · Thermo Fisher — Alexa Fluor 488 Protein Labeling Kit manual (ε and correction factor)

Frequently asked questions