Protein labeling calculator

Calculate the dye amount for NHS ester fluorescent dye conjugation, and determine the measured degree of labeling (DOL) from UV-Vis absorbance

Input parameters

Protein MW (Da)

Dye MW (Da)

Reference: Cy5-NHS ≈ 792, FITC ≈ 389

Protein concentration (mg/mL)

Protein volume (μL)

Dye stock concentration (mM)

Typically 10 mM in DMSO

Dye : protein molar ratio

Recommended 5–20×

Results

Fill in the parameters and click "Calculate" to see results

Measured degree of labeling (DOL)

DOL cannot be predicted from the molar excess used; it is measured from the absorbance spectrum after the reaction, once free dye has been removed. Fill in the five fields below. Path length and dilution cancel out in this ratio, so they are not required.

A₂₈₀ (absorbance at 280 nm)

A_max (absorbance at the dye λmax)

Dye extinction coefficient ε (M⁻¹cm⁻¹)

See the product page or CoA; reference: AF 488 ≈ 73000, Cy5 ≈ 250000

Dye correction factor CF₂₈₀

The dye's absorbance at 280 nm as a fraction of its λmax, from the dye datasheet; entering 0 makes the DOL read low

Protein extinction coefficient ε (M⁻¹cm⁻¹)

IgG reference value 203000; use the actual value for other proteins

Fill in all five fields on the left to calculate the DOL

Calculation principles

Protein amount (μmol) = concentration (mg/mL) × volume (μL) / MW (Da)

Dye required (μmol) = protein amount (μmol) × molar ratio

Dye stock volume (μL) = dye required (μmol) / stock concentration (mM) × 1000

Measured DOL = (A_max × ε_protein) / ((A₂₈₀ − A_max × CF₂₈₀) × ε_dye)

Note: the molar excess only determines how much dye to add — it is not the degree of labeling. The actual DOL is affected by buffer pH, reaction time, protein activity and other factors. Use the UV-Vis measured value as the definitive result.