This calculator estimates the protein extinction coefficient at 280 nm using aromatic residue content and disulfide bonds.
Molar extinction coefficient at 280 nm:
ε280 = (5500 × nW) + (1490 × nY) + (125 × nSS)
Beer–Lambert law:
A = ε × c × l
Specific mass coefficient: εmass = ε280 / MW
Here, nW is the number of tryptophan residues, nY is the number of tyrosine residues, and nSS is the number of disulfide bonds. A is absorbance, c is concentration, and l is path length.
The specific mass coefficient in L g-1 cm-1 is numerically equal to mL mg-1 cm-1, which helps concentration work in mg/mL.
- Choose sequence mode for automatic counting, or manual mode for direct residue entry.
- Enter the protein sequence or manual counts for tryptophan, tyrosine, and cysteine.
- Provide the number of disulfide bonds actually present.
- Enter path length, and add molecular weight if you already know it.
- Add measured A280 to estimate concentration from Beer–Lambert law.
- Add known concentration to predict absorbance and compare measured versus expected values.
- Review the result cards, table summary, and Plotly graph.
- Use the CSV or PDF buttons to export the final report.
| Sample | Trp (W) | Tyr (Y) | Disulfides | Molecular Weight (Da) | ε280 (M^-1 cm^-1) | Specific Coefficient (mL mg^-1 cm^-1) |
|---|---|---|---|---|---|---|
| Example Sample A | 2 | 4 | 1 | 25,000 | 17,085 | 0.683400 |
| Example Sample B | 1 | 8 | 2 | 66,000 | 17,670 | 0.267727 |
| Example Sample C | 0 | 6 | 3 | 14,000 | 9,315 | 0.665357 |
1. What is a protein extinction coefficient?
It measures how strongly a protein absorbs ultraviolet light, usually at 280 nm. It helps estimate concentration from absorbance data.
2. Why is 280 nm commonly used?
Proteins absorb near 280 nm mainly because tryptophan and tyrosine residues absorb strongly there. Disulfide bonds make a smaller contribution.
3. Do all cysteines increase the extinction coefficient?
No. The standard estimate adds the cysteine term only for disulfide-linked cystines, not for every free cysteine residue.
4. What happens if my sequence has no tryptophan?
The coefficient can still be nonzero if tyrosines or disulfide bonds are present. If none are present, the predicted A280 may be very low.
5. Is molecular weight required?
It is required for the mass-specific coefficient and mg/mL concentration estimate. In sequence mode, this tool can estimate molecular weight automatically.
6. Which concentration units does the calculator support?
It reports molar concentration in mol/L and mass concentration in mg/mL when enough information is available.
7. Can I use absorbance to estimate protein concentration?
Yes. Enter A280 and path length. If extinction coefficient and molecular weight are known or estimated, the calculator returns concentration.
8. Is this result exact for every protein?
No. It is a practical estimate. Buffer composition, folding state, chromophores, contaminants, and experimental conditions can shift real absorbance values.