Protein Extinction Coefficient Calculator
Calculate molar extinction coefficient and protein concentration from amino acid composition.
Where the Coefficients Come From
Protein absorbance at 280 nm comes almost entirely from three species, and the Pace method sums their individual contributions.
| Residue | ε at 280 nm | Contribution |
|---|---|---|
| Tryptophan | 5,500 | Dominant |
| Tyrosine | 1,490 | Secondary |
| Cystine (disulfide) | 125 | Minor |
| Phenylalanine | ~0 at 280 | Absorbs at 257 nm |
Tryptophan contributes nearly four times as much as tyrosine per residue, so a protein lacking tryptophan has a low extinction coefficient and A280 becomes an unreliable way to measure it. Some proteins have no tryptophan or tyrosine at all and are effectively invisible at 280 nm.
Note that cystine, not cysteine, contributes. Free thiols absorb negligibly, so the coefficient differs between reduced and oxidised forms of the same protein — typically a 2–5% difference.
Accuracy and Its Limits
The Pace method is accurate to roughly 5% for folded proteins and rather better for denatured ones, because the local environment of aromatic residues shifts their absorbance slightly. Buried tryptophans absorb differently from exposed ones.
| Interference | Effect | Handling |
|---|---|---|
| Nucleic acid | Strongly inflates A280 | Check A260/A280 ratio |
| Scattering from aggregates | Raises apparent absorbance | Subtract A320 baseline |
| Haem or other cofactors | Absorb at 280 | Use a cofactor-corrected coefficient |
The A260/A280 ratio is the standard purity check. Pure protein gives about 0.57 and pure DNA about 1.8 — so even a few percent nucleic acid contamination raises the ratio noticeably and inflates the apparent protein concentration.
Turbidity from aggregates scatters light across the spectrum. Since protein does not absorb at 320 nm, any reading there indicates scattering and should be subtracted from A280 before calculating concentration.
Worked Examples
Common Mistakes
Tryptophan dominates the coefficient. Proteins lacking it have low ε and poor sensitivity; some have none and are invisible at 280 nm.
Nucleic acids absorb strongly at 280 and inflate apparent protein concentration. Check the A260/A280 ratio, which should be near 0.57 for pure protein.
Only disulfide-bonded cystine contributes. The coefficient differs between reduced and oxidised forms of the same protein.
Aggregates scatter light and raise apparent absorbance. Protein does not absorb at 320 nm, so any signal there should be subtracted.