Protein Concentration (A280) Calculator
The concentration of a purified protein from its absorbance at 280 nm, its molar extinction coefficient and molecular weight — in mg/mL and micromolar, with the total amount in your sample.
Tryptophan, tyrosine and cystine absorb at 280 nm, so a protein’s absorbance there is proportional to its concentration by Beer–Lambert: A = ε × c × l.
How the protein concentration (a280) calculator works
Tryptophan, tyrosine and cystine absorb at 280 nm, so a protein’s absorbance there is proportional to its concentration by Beer–Lambert: A = ε × c × l. The molar extinction coefficient is computed from the sequence (ProtParam) or taken from the literature — 43,824 M⁻¹cm⁻¹ for BSA. Divide by ε for molarity, multiply by the molecular weight for mg/mL. It is only accurate for a pure protein with no nucleic acid contamination.
Formula: c (M) = A₂₈₀ × dilution ÷ (ε × l); mg/mL = c × MW ÷ 1,000
Worked examples
| Inputs | Concentration | Note |
|---|---|---|
| BSA, A₂₈₀ 0.66 | 0.001 mg/mL | 1.0 mg/mL, 15 µM |
| Lysozyme, A₂₈₀ 0.5 at 1:5 | 0.001 mg/mL | 0.94 mg/mL |
| IgG, A₂₈₀ 1.2 | 0.001 mg/mL | 0.86 mg/mL |
FAQFrequently asked questions
Where do I get the extinction coefficient?
From the sequence: ExPASy ProtParam sums 5,500 per tryptophan, 1,490 per tyrosine and 125 per cystine. Literature values exist for common proteins; a protein with no Trp or Tyr cannot be measured this way.
Why not use the 1 Abs = 1 mg/mL rule?
Because the mass extinction coefficient ranges from about 0.3 to 3 (mg/mL)⁻¹cm⁻¹ between proteins; the rule can be off by a factor of two or three. The calculator shows the true 0.1% coefficient for your protein.
What about nucleic acid contamination?
DNA and RNA absorb strongly at 260 and 280; an A260/A280 above about 0.7 suggests contamination, which inflates the protein figure. The Warburg–Christian correction or a dye assay (Bradford, BCA) is more reliable then.
Does buffer matter?
Yes — blank against the same buffer, and avoid components that absorb at 280 (DTT, some detergents, imidazole above a few mM). Reducing agents oxidise and their absorbance drifts.
Where these figures come from
- NCBI Bookshelf — Molecular Biology of the Cell — growth kinetics and molecular conventions
- Wellcome Sanger Institute — a leading UK genomics institute
Last checked: September 2026. Formulas are the standard textbook forms; assumptions are stated on each page because they are where these models break.