A280 protein concentration calculator
Concentration in mg/mL and µM from absorbance at 280 nm, with ε from a value or from the sequence.
Concentration in mg/mL and µM from absorbance at 280 nm, with ε from a value or from the sequence.
Proteins absorb ultraviolet light at 280 nm because of their tryptophan and tyrosine residues, and to a small extent their disulfide bonds. The Beer–Lambert law turns the absorbance reading into a concentration:
A280 is the blank-corrected absorbance, ε is the molar extinction coefficient in M⁻¹cm⁻¹, l is the light path in cm and MW the molecular weight in g/mol. Enter ε directly on the first tab, or paste the sequence on the second tab and the calculator derives ε and the molecular weight for you.
Worked example. BSA read at A280 = 0.66 in a 1 cm cuvette with ε = 43,824 M⁻¹cm⁻¹: 0.66 ÷ 43,824 = 1.51 × 10⁻⁵ M = 15.1 µM. Times 66,463 g/mol gives 1.00 mg/mL.
For a protein in water the coefficient is the sum of its chromophores, following Pace and co-workers (1995):
Cystine means a disulfide bond, so the last term counts pairs of cysteines. TheFrom sequence tab reports both the value with all cysteines paired and the fully reduced value, and uses the paired value for the concentration, which is the usual assumption for a native protein. Some common values:
| Protein | ε280 (M⁻¹cm⁻¹) | MW (Da) | A280 at 1 mg/mL |
|---|---|---|---|
| Bovine serum albumin | 43,824 | 66,463 | 0.66 |
| Human IgG | ~210,000 | ~150,000 | ~1.4 |
| Hen lysozyme | 37,970 | 14,313 | 2.65 |
| Ovalbumin | 30,590 | 42,750 | 0.72 |
The last column, the absorbance of a 0.1% (1 mg/mL) solution, equals ε ÷ MW × 10 and is what datasheets often quote. The result panel shows it for your protein. For a coefficient at other wavelengths or for peptides see theextinction coefficient calculator.
Only as a rough rule. It holds for an average protein, but the real figure spans roughly 0.5 to 2.5 mg/mL per absorbance unit: BSA is 1.5 mg/mL per unit, lysozyme only 0.38. A protein with no tryptophan or tyrosine, such as some small peptides, barely absorbs at 280 nm and needs a colorimetric assay instead.
Cuvette readings use a 1 cm path. NanoDrop and similar microvolume instruments read through 0.05 to 1 mm but report the value normalised to 10 mm, so enter them with a path length of 1 cm. If you diluted the sample before reading, enter the fold dilution and the calculator scales the result back to the original stock. For accuracy keep the measured absorbance between about 0.1 and 1.0, diluting if necessary, and blank with the same buffer the protein is in.
Nucleic acids absorb about ten times more strongly at 280 nm than protein does per microgram, so a small DNA or RNA contamination gives a large overestimate. A pure protein has an A260/A280 ratio near 0.57; a rising ratio flags contamination. The classic Warburg–Christian correction estimates protein in a mixed sample as:
Better still is a nuclease treatment or a further purification step before measuring.
Use the Beer–Lambert law. Molar concentration = A280 × dilution factor ÷ (ε × path length), where ε is the molar extinction coefficient at 280 nm in M⁻¹cm⁻¹ and the path length is in cm. Multiply by the molecular weight in g/mol to convert to g/L, which is the same as mg/mL.
Only roughly. A280 of 1.0 corresponds to about 1 mg/mL for an average protein, but the true value ranges from about 0.5 to over 2 mg/mL depending on tryptophan and tyrosine content. BSA at 1 mg/mL reads 0.66, lysozyme reads about 2.6. Use the protein's own extinction coefficient.
It is the sum of the contributions of its chromophores: 5,500 M⁻¹cm⁻¹ per tryptophan, 1,490 per tyrosine and 125 per cystine disulfide bond. The From sequence tab computes it from the amino acid sequence. A protein with no Trp or Tyr cannot be measured at 280 nm.
NanoDrop instruments report absorbance already normalised to a 10 mm path, so enter the reading with a path length of 1 cm. If your instrument reports the raw 1 mm reading, enter 0.1 cm. Keep readings between about 0.1 and 1.0 absorbance units for accuracy.
DNA and RNA absorb strongly at 280 nm and inflate the result. Check the A260/A280 ratio: pure protein gives about 0.57, pure nucleic acid about 2.0. For mixed samples use the Warburg–Christian correction, protein (mg/mL) ≈ 1.55 × A280 − 0.76 × A260, or purify the sample before measuring.