Protein Concentration from A280 Calculator

Understanding the protein concentration from A280 calculator — concept and principle

Measuring absorbance at 280 nm is the quickest way to find the concentration of a purified protein: it needs no reagents and the sample can be recovered. This calculator applies the Beer-Lambert law to an A280 reading, using the protein's molar extinction coefficient and molecular weight, and returns the concentration in mg/mL and µM. The defaults are for bovine serum albumin (BSA).

A280 0.8 in a 1 cm cell gives 18.25 µM, or 1.213 mg/mL of BSA.

How it is calculated

Molar concentration: c (M) = A ÷ (ε × l).
µM: c × 10⁶.
mg/mL: c × MW, because mol/L × g/mol = g/L, and 1 g/L = 1 mg/mL.

A280 is dimensionless, ε is in M⁻¹cm⁻¹, l is the light path in cm (1 cm for a standard cuvette), and MW is in Da (g/mol).

The principle

The Beer-Lambert law states that absorbance is proportional to the concentration of the absorbing species and to the distance light travels through it: A = εcl. At 280 nm, almost all protein absorbance comes from the aromatic side chains of tryptophan and tyrosine, with a small contribution from disulfide bonds. Each protein therefore has its own ε, which can be measured or predicted from the amino-acid sequence. Rearranging the law gives c = A ÷ (εl).

Worked example

Inputs: A280 = 0.8, ε = 43,824 M⁻¹cm⁻¹, MW = 66,430 Da, l = 1 cm.
c = 0.8 ÷ (43,824 × 1) = 1.8255 × 10⁻⁵ M = 18.25 µM.
Mass concentration: 1.8255 × 10⁻⁵ mol/L × 66,430 g/mol = 1.2127 g/L = 1.213 mg/mL.

Each point is the calculator re-run with only “Extinction coefficient (M⁻¹cm⁻¹)” changed; the gold point is the default example. Change the inputs above and this chart follows your numbers.

Extinction coefficient (M⁻¹cm⁻¹)	Concentration (mg/mL)
10,956	4.850675429
21,912	2.425337714
32,868	1.61689181
43,824	1.212668857
54,780	0.9701350858
65,736	0.8084459048
87,648	0.6063344286

How to read the result

ε ÷ MW gives the absorbance of a 1 mg/mL solution; for these BSA values it is about 0.66, so an A280 of 0.8 corresponds to about 1.2 mg/mL. Proteins rich in tryptophan have much higher values. Readings in the middle of the instrument's range are the most reliable; dilute samples that read too high and use a shorter path for concentrated ones. The A260/A280 ratio helps detect nucleic-acid contamination, which absorbs strongly at 260 nm and inflates A280.

Common mistakes

Forgetting to blank the instrument with the same buffer the protein is in.
Using ε for the reduced protein when disulfides are formed, or the reverse.
Measuring cloudy samples; light scattering raises apparent absorbance.
Forgetting the dilution factor when the sample was diluted before reading.

Keep in mind: The result is only as accurate as ε. Predicted coefficients are usually within a few percent for proteins with several Trp and Tyr residues, but much less reliable for proteins with few or none, where colourimetric assays may be better.

Frequently asked questions

Where do I get the extinction coefficient?

Calculate it from the sequence using the Pace method or a tool such as ExPASy ProtParam, or use a published experimental value.

What is "A280 0.1%"?

The absorbance of a 1 mg/mL (0.1% w/v) solution in a 1 cm path, equal to ε ÷ MW.

Can I use this for a protein mixture or cell lysate?

Not reliably, because the mixture has no single ε. Use a Bradford or BCA assay instead.

Sources: C. N. Pace, F. Vajdos, L. Fee, G. Grimsley and T. Gray, Protein Science 4, 2411–2423 (1995) · E. Gasteiger et al., "Protein identification and analysis tools on the ExPASy server", The Proteomics Protocols Handbook, Humana Press (2005)

Checked 8 Nov 2026. Standard textbook formulas in SI units, computed in your browser. For learning and estimates — real designs must follow your local codes and be checked by a qualified engineer.

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