Protein DOL Calculator

How This Calculator Works

Three measurements from your labeled protein sample — A₂₈₀, A_dye, and dilution factor — are all you need.

1 Correct for dye bleed-in

Most dyes absorb weakly at the wavelength used to measure protein concentration, 280nm. Left uncorrected, this inflates your protein reading. A correction factor (CF₂₈₀, specific to each dye) is used to remove the dye's contribution from A₂₈₀.

A₂₈₀ corrected = A₂₈₀ − (A_dye × CF₂₈₀)

2 Calculate protein concentration

The corrected A₂₈₀ is divided by the protein's extinction coefficient to get concentration in mg/mL. If you diluted your sample before reading, multiply by the dilution factor to recover the original stock concentration. Protein molarity is then calculated by dividing by molecular weight.

Conc = (A₂₈₀ corrected / E_0.1%) × dilution
Molarity = (A₂₈₀ corrected / E_0.1%) / MW

3 Calculate dye molarity and DOL

Dye concentration is calculated using the A_max of the dye and divided by the dye's molar extinction coefficient to give dye molarity. Dividing dye molarity by protein molarity gives the degree of labeling: the average number of dye molecules attached per protein.

Dye molarity = A_dye / ε_dye
DOL = dye molarity / protein molarity

Extinction coefficient modes: Most preset proteins use E(0.1%) — absorbance of a 1 mg/mL solution. Custom proteins can use either E(0.1%) or a molar extinction coefficient (ε). If you enter ε, protein molarity is calculated first, then concentration is derived from it.

Dilution factor tip: Dilute your sample if A₂₈₀ or A_dye exceeds ~1.0. Enter the dilution factor (e.g. 5 for a 1:5 dilution) and the calculator will report the concentration of your original stock. The dilution factor does not affect DOL, since it cancels out in the ratio.

Parameters

Select Protein

Extinction Type

Protein MW (Da)

Protein E (0.1%)

Select Dye / Label

Dye ε (Molar)

Dye CF₂₈₀

A₂₈₀

A_dye

Note: The linear range of most spectrophotometers is between 0.1 and 1.5 absorbance units (AU). Check the documentation for your instrument to determine the linear range of the detector. If your readings fall outside this range, dilute your sample and remeasure.

Dilution Factor