How to use
- Enter the A280 you measured, already blanked against your buffer.
- Leave the path length at 1 cm unless you used a shorter cell or a microvolume instrument, which usually reports a 1 cm equivalent anyway.
- Say which kind of coefficient you have and enter it. A molar coefficient also needs the molecular weight.
- No coefficient? Fill in the residue counts underneath and the page works one out from the sequence.
💡 Exponents can be entered with e — for example, 1.5×10⁻⁵ is entered as 1.5e-5.
Formula & notes
Beer-Lambert, A = ε·c·l, rearranged for the concentration. Everything on this page is that one line; the difficulty is entirely in ε, which is a property of your protein and not a number anyone can supply for you.
Three ways in, one calculation.
- From a molar coefficient
- c (mol/L) = A ÷ (ε × l) then mg/mL = c × MW
- From A(1 %, 1 cm)
- c (mg/mL) = A ÷ (A(1 %) ÷ 10 × l)
- From ε0.1 %
- c (mg/mL) = A ÷ (ε0.1 % × l)
The residue contributions
| Residue | Contribution to ε₂₈₀ | Note |
|---|---|---|
| Tryptophan | 5500 M⁻¹cm⁻¹ | dominates the total in most proteins |
| Tyrosine | 1490 M⁻¹cm⁻¹ | |
| Cystine | 125 M⁻¹cm⁻¹ | per disulfide bond, not per cysteine |
The three coefficient conventions describe the same absorbance from different reference concentrations: ε is per mole, A(1 %) is the absorbance of a 10 mg/mL solution, and ε0.1 % that of a 1 mg/mL solution. A(1 %) divided by ten is ε0.1 %, which is why the tool asks which one you have rather than guessing from the magnitude.
Practical notes
- ⭐ This tool ships no default extinction coefficient, and that is a decision rather than an omission. "A280 of 1 is about 1 mg/mL" is repeated everywhere and there is no primary source that makes it true for your protein. This site does not print an unsourced number where a criterion goes; it has declined to draw a pass mark on western blot CV and on transformation efficiency for the same reason.
- The sourced way to get ε is from the composition: ε₂₈₀ = 5500·n(Trp) + 1490·n(Tyr) + 125·n(cystine). Pace CN, Vajdos F, Fee L, Grimsley G, Gray T (1995), How to measure and predict the molar absorption coefficient of a protein, Protein Sci 4(11):2411-2423, fitted to 116 measured values across 80 proteins.
- ⚠️ The paper's own limitation travels with it. The prediction is reliable for proteins containing tryptophan and less reliable for those without, and it is for the folded protein in water. In 6 M guanidinium chloride the residue contributions are different.
- ⚠️ Cystine is the disulfide pair, not the number of cysteines. Counting free cysteines roughly doubles that term. It is the one input on this page a reader will misread, which is why the label spells it out.
- Keep the reading in the linear range — roughly 0.1 to 1.0 on a 1 cm path. Above that a spectrophotometer stops being linear and the number it gives is not an absorbance any more; dilute and multiply back.
- Nucleic acid absorbs at 280 too. If A260/A280 is well above 0.6 for a pure protein, the reading is inflated and this conversion will over-report the concentration.
FAQs
Often used together
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Molarity ↔ mg/mL Converter
Converts between molarity and mass concentration through the molecular weight, with no stray factor of 1000.