Protein labeling calculator

Calculate measured DOL, protein concentration and dye input amounts.

Target protein

Labeling dye

A₂₈₀ · 280 nm

Amax · Dye absorption peak

Remove free dye first. Both readings must use the same sample, effective path length and dilution.

Measurement settings and parametersPath 1 cm · Dilution 1×

Measurement conditions

Path length of readingscm

Enter 1 for absorbance normalized to 1 cm; otherwise use the actual optical path.

Dilution factor×

Use 1 for undiluted samples, 10 for tenfold dilution. Use 1 if readings already account for dilution.

Reference parameters

IgG reference values

Protein extinction coefficient εM⁻¹·cm⁻¹

Use the target protein’s molar extinction coefficient at 280 nm.

Protein molecular weightDa

Use the unlabeled protein mass. For oligomers, match the molecular basis of the selected preset.

Dye extinction coefficient εM⁻¹·cm⁻¹

Use the dye’s molar extinction coefficient at its absorption peak.

280 nm correction factor CF₂₈₀

The ratio of dye absorbance at 280 nm to its absorption peak. Check the manual if unknown; do not substitute zero.

IgG: ε = 203,000 from the labeling manual, with a reference intact-IgG mass of 150,000 Da. Use antibody-specific documentation when available.

FluoReporter FITC F6434, MP06434, pp. 3–4

NanoDrop Protein A280 technical guide, p. 15

Custom parameters: use the relevant manual, CoA or protein documentation.

Principles and common questions

How does the input molar ratio differ from DOL?

The input molar ratio determines how much dye to add before the reaction. DOL is measured after purification; it cannot be directly inferred from the input ratio.

Which results depend on path length and dilution?

They cancel in DOL when both absorbance readings use the same sample, effective path length and dilution. Protein concentration still needs both values. For microvolume readings normalized to 1 cm, enter 1 cm; apply dilution correction only once.

What if my dye is not listed?

Choose custom and enter ε and CF₂₈₀ from your product documentation. Qihe SF manuals lack both values; Qihe Cyanine manuals lack CF₂₈₀, so automatic presets are not provided yet. Similar wavelengths, family names or other suppliers do not justify substituting parameters.

Which samples can use this method?

Use purified protein without free dye, other proteins or significant absorbing contaminants. cSA is counted per tetramer. Colored proteins such as PE, APC and PerCP may need bidirectional spectral correction, and oligonucleotides typically use A₂₆₀; they should not use this calculation directly.

The optimal DOL depends on the protein, dye and application. Validate experimentally.