Protein analysisBeer–Lambert law

A280 Protein Concentration Calculator

Calculate protein concentration from A280 using a known molar extinction coefficient or derive ε280 and average molecular weight from sequence.

Runs locally in your browser No intermediate rounding
Measurement inputs
Enter a known ε280 or estimate ε280 and average molecular weight from a protein sequence.
Extinction coefficient source
Absorbance reading at 280 nm
Use 0 if already blank-corrected
Use the value for your protein at 280 nm
M⁻¹ cm⁻¹
Use the instrument’s effective path
cm
Undiluted sample = 1
Needed only to calculate mg/mL
Your sequence, measurements, and calculated values stay in this browser and are not included in analytics.
Calculated concentration
Blank-corrected and adjusted to the original sample.

Molar concentration

20 µM

2.00000e-5 M

Mass concentration

1 mg/mL

Measured A280

1

Blank A280

0

Corrected A280

1

Dilution factor

1×

Calculation used

c = (A corrected × DF) / (ε × l)
c = (1 × 1) / (50,000 × 1)
c = 2.000000e-5 M

Check the method assumptions

This result assumes the entered ε280 applies to the protein state and buffer used, and that no other chromophore or prosthetic group materially absorbs at 280 nm.

Buffer blanking, path length, dilution, protein state, and instrument behavior can affect the result. Conjugated or labeled proteins may need a different correction workflow.

Method

How A280 concentration is calculated

The calculator first subtracts the blank from the measured A280, then applies Beer–Lambert law. Dilution factor restores the concentration of the original, undiluted sample.

A corrected = A measured − A blank
concentration (M) = (A corrected × dilution factor) / (ε × path length)
concentration (mg/mL) = concentration (M) × molecular weight (g/mol)

mg/mL is numerically equal to g/L. The calculator retains full precision internally and rounds only the displayed result.

Before using A280

A280 works best for purified proteins with a reliable extinction coefficient. Estimates derived from sequence are generally less reliable for proteins without tryptophan.

A sequence-derived ε280 is an estimate, not an instrument calibration. It depends on assumptions about cysteine oxidation and the protein’s absorbing groups.

This calculator does not assess A260/A280 purity and is not a BCA or Bradford standard-curve calculator.

Choosing an extinction coefficient

Use a measured or trusted molar ε280 for the same protein form whenever possible. Molar coefficients use M⁻¹ cm⁻¹; do not enter a mass extinction coefficient in its place.

Sequence estimates commonly use contributions from tryptophan, tyrosine, and cystine. Cystine means disulfide bonds—not the raw number of cysteine residues.

What if my instrument already corrects for path length?

Enter the effective path length that corresponds to the reported absorbance. For a reading normalized to a 1 cm path, use 1 cm. Consult the instrument method when it reports concentration or applies a proprietary correction directly.

Can corrected A280 be zero?

Yes. A measured value equal to the blank produces zero concentration. If the blank is higher than the measurement, the calculator reports a validation warning instead of a negative concentration.

Why is molecular weight optional?

Beer–Lambert law returns molar concentration without molecular weight. Molecular weight is needed only to convert the molar result to mg/mL.

Continue your workflow

Convert mg/mL and molarity

Convert a known protein concentration across mass and molar units.

Open calculator

Calculate molecular weight from sequence

Inspect average and monoisotopic mass with the same shared sequence engine used by this A280 calculator.

Open calculator
Scientific basis: ExPASy ProtParam documentation and Pace et al. (1995), PMID 8563639. This browser tool supports laboratory planning and does not replace method validation.