A280 Protein Concentration Calculator

Calculate the concentration of a protein sample from an A280 measurement using the Beer–Lambert relationship.

Measurement and Protein Inputs

Measured A280Absorbance is dimensionless, although instruments may display it in absorbance units (AU).Blank A280Use 0 if the instrument already blank-corrected the measurement.Molar extinction coefficient, ε280 (M⁻¹ cm⁻¹)Molar extinction coefficient is the common laboratory term; molar absorption coefficient is the more formal term.Effective path length (cm)Dilution factorUse 1 for an undiluted sample.Protein molecular mass (kDa) (optional)Needed to report mass concentration; leave blank for molar concentration only.

The Beer–Lambert proportionality is an approximation that works best for clear, homogeneous samples measured within the instrument’s validated linear absorbance range. Use a buffer-matched blank and the effective path length reported by the instrument. A280 estimates can be affected by nucleic-acid contamination, scattering or turbidity, absorbing cofactors, aggregation, and other sample impurities.

How A280 protein concentration is calculated

The calculator uses the Beer–Lambert relationship: corrected A280 is divided by the product of ε280 and path length to give molar concentration. The dilution factor then restores the concentration of the original sample rather than the diluted aliquot that was measured.

Why subtract a blank?

A buffer-matched blank corrects for signal from the buffer, cuvette, and instrument baseline. The remaining A280 may still contain contributions from contaminants, absorbing cofactors, and light scattering in addition to protein absorption.

When is molecular mass needed?

ε280 gives molar concentration directly. Molecular mass is needed only to express that molar concentration as mass concentration, such as mg/mL or µg/µL.

Choosing a theoretical ε280

Sequence-derived ε280 estimates depend on whether cysteines are reduced or use the maximum cystine-pair assumption. The latter represents the maximum number of cystine pairs possible from the sequence, so it may not match the measured protein’s actual disulfide state.

When not to trust a simple A280 estimate

Treat the result cautiously for impure samples, substantial nucleic-acid contamination, scattering or turbidity, an unsuitable blank, an unknown effective path length, proteins with very low ε280, or samples containing absorbing cofactors. These situations need a method designed for the relevant interference rather than a simple A280 calculation.