How to Estimate Protein Concentration from A280

What an A280 concentration estimate measures

An A280 measurement records attenuation of light near 280 nm. Proteins commonly absorb at this wavelength because of aromatic residues, especially tryptophan and tyrosine, and because cystine can contribute when cysteines are paired. Given an appropriate ε280 value, the measurement can estimate molar protein concentration.

It is an optical estimate, not a protein-specific count. The remaining signal after blank correction can include contributions from nucleic acids, cofactors, turbidity, aggregates, and other sample components. Treat the result as most reliable for a clear, homogeneous sample with a suitable blank and a validated measurement range.

The Beer–Lambert relationship used by the calculator

The calculator first subtracts the blank, then applies the Beer–Lambert relationship and any dilution correction:

Absorbance is dimensionless, although instruments may display it in absorbance units (AU). The dilution factor is 1 for an undiluted sample. If a diluted aliquot was measured, multiplying by its dilution factor reports the concentration of the original sample rather than the concentration in the cuvette or pedestal at the time of reading.

Why use a buffer-matched 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. A blank therefore improves the measurement; it does not make the result protein-specific.

Use the effective path length reported by the instrument, rather than assuming a 1 cm path length for every measurement format. Microvolume instruments and plate measurements can use an effective path length different from a conventional cuvette.

What ε280 means

ε280 is the molar extinction coefficient at 280 nm, expressed in M⁻¹ cm⁻¹. It links absorbance to molar concentration for a particular protein under the stated assumptions. Molar extinction coefficient is the common laboratory term; molar absorption coefficient is the more formal term.

A sequence-derived ε280 is useful when a reliable experimental value is unavailable, but it remains theoretical. Tags, cleavage, post-translational modifications, bound cofactors, changes in the measured protein species, and contaminants can make an experimentally observed A280 differ from the sequence-only estimate.

Using sequence-derived ε280 values

The Protein Properties Calculator reports two theoretical ε280 values that can be transferred directly to the A280 calculator:

• Reduced cysteines: the sequence-derived value without a cystine contribution.
• Maximum cystine-pair assumption: the sequence-derived value that includes the maximum number of cystine pairs possible from the sequence.

The maximum-cystine-pair value is not a prediction that every cysteine participates in a disulfide bond. For an odd number of cysteines, one cysteine necessarily remains unpaired. Choose the assumption that best reflects the actual protein and experimental context, rather than treating either theoretical value as a confirmed oxidation state.

Worked example: a 50 kDa protein

Suppose the measured diluted sample has the following values:

• Measured A280: 0.65
• Blank A280: 0.05
• ε280: 50,000 M⁻¹ cm⁻¹
• Path length: 1 cm
• Dilution factor: 10
• Protein molecular mass: 50 kDa

The same mass concentration is also 6 µg/µL. You can enter these values in the A280 Protein Concentration Calculator, then continue directly to the Protein Molarity Calculator for mass, mole, molecule-count, and concentration conversions.

When molecular mass is needed

Molecular mass is not needed to calculate molar concentration from ε280. It is needed to express that result as mass concentration. Multiplying molar concentration in mol/L by molar mass in g/mol gives g/L, which is numerically equal to mg/mL. This is why a molecular mass of 50 kDa converts the 120 µM result above to 6 mg/mL.

Use the molecular mass of the measured species: for example, the tagged construct, mature processed protein, monomer, or intact complex that the concentration represents. A sequence-derived value may need adjustment for modifications or a different species.

When not to trust a simple A280 estimate

The Beer–Lambert proportionality is an approximation that works best for clear, homogeneous samples measured within the instrument's validated linear absorbance range. Treat a simple A280 estimate cautiously when any of the following apply:

• Substantial nucleic-acid contamination contributes absorbance near 280 nm.
• Scattering or turbidity from particles, precipitation, or aggregation affects transmission.
• The blank is not buffer-matched, or the effective path length is unknown.
• The protein has a very low ε280, making small background effects comparatively important.
• Absorbing cofactors, ligands, dyes, or other impurities contribute at or near 280 nm.
• The measurement falls outside the instrument's validated linear absorbance range.

In these cases, investigate the source of the interference and use a method suited to the sample and question. Do not interpret an A280 result as an independently verified pure-protein concentration when the measurement conditions do not support that claim.

References

• Mayerhöfer T. G., Pahlow S., and Popp J. The Bouguer–Beer–Lambert Law: Shining Light on the Obscure. ChemPhysChem, 2020.
• Protein Properties Guide for the sequence-derived ε280 assumptions used by Calcorium.