Inputs
Formula used
This tool uses a common 280 nm model for proteins:
- Extinction coefficient estimate: ε280 = 5500·NW + 1490·NY + 125·NSS
- Beer–Lambert law: A = ε·c·l, so c = A / (ε·l)
- Optional correction: A280,corr = A280 − 0.57·A260
Here NW is tryptophan count, NY is tyrosine count, and NSS is the number of disulfide bonds (cystine pairs).
How to use this calculator
- Paste a one-letter protein sequence, or enter W/Y/C counts manually.
- Choose the cysteine state and optionally set disulfide bonds.
- Enter A280 and the path length; add dilution factor if used.
- Enable A260 correction only when A260 is available.
- Provide molecular weight to also get concentration in mg/mL.
- Press Calculate to show results above, then export if needed.
Example data table
| Example | W | Y | C | Disulfides | A280 | Path (cm) | Dilution | ε280 (M^-1 cm^-1) | c (uM) |
|---|---|---|---|---|---|---|---|---|---|
| Sample A | 2 | 5 | 4 | 2 | 0.85 | 1.0 | 10 | 18,700 | 454 |
| Sample B | 1 | 3 | 0 | 0 | 0.42 | 0.5 | 1 | 9,970 | 84.2 |
Professional article
1) Why extinction coefficient matters
The 280 nm extinction coefficient ε280 links absorbance to molar concentration, enabling fast protein quantification without colorimetric reagents. Reliable ε supports dosing and kinetics. This calculator estimates ε from aromatic residues and converts A280 to concentration with path-length and dilution handling.
2) Sequence-based estimation at 280 nm
At 280 nm, tryptophan and tyrosine dominate protein absorbance. The estimator uses ε280 = 5500·NW + 1490·NY plus an optional disulfide term. When you paste a one-letter sequence, the tool counts W and Y automatically, making the estimate traceable across runs.
3) Cysteine chemistry and disulfide contribution
Cysteine’s contribution depends on oxidation. When cysteines form disulfide bonds (cystine), the model adds 125 per bond. Select oxidized to include this term and either provide disulfide count or let the tool approximate floor(C/2). Select reduced when thiols are free or chemically reduced.
4) Absorbance inputs and path length
Absorbance scales with optical path length l. Standard cuvettes use 1 cm, while microvolume devices may use 0.05–1.0 cm. Enter the instrument’s reported path length and the measured A280. If you diluted the sample, enter the dilution factor so the output reflects the original stock concentration.
5) Beer–Lambert conversion and units
Beer–Lambert law states A = ε·c·l, so c = A/(ε·l). The calculator reports c in molar units and µM for convenience. If you provide molecular weight, it also reports mg/mL by converting c·MW to g/L, which is numerically equal to mg/mL.
6) Handling nucleic-acid contamination
Nucleic acids absorb strongly at 260 nm and can inflate A280-based protein estimates. When A260 is available, you can apply the correction A280,corr = A280 − 0.57·A260. Use corrected results as a screening value; mixtures, scattering, or unusual chromophores can still bias absorbance-based calculations.
7) Reasonableness checks and uncertainty
Quality checks help catch bad reads. Very high A values can indicate bubbles, fingerprints, or saturation; remeasure with dilution if needed. Compare estimated ε with literature values, and override with a verified manual ε when accuracy matters. Keep units consistent: ε in M−1·cm−1 and l in cm.
8) Reporting, documentation, and exports
For documentation, the results table records residue counts, disulfide assumption, ε estimated and used, and concentration in multiple units. Export buttons generate a CSV for spreadsheets and a compact PDF for lab notebooks. Saving parameters alongside results clearly improves reproducibility and simplifies review during QC or troubleshooting.
FAQs
1) Should I paste a sequence or enter residue counts?
If you have the sequence, paste it. Automatic counting reduces mistakes and ensures W/Y/C totals match the protein. Use manual counts when the sequence is unknown or deliberately modified.
2) What does the “oxidized” cysteine option change?
Oxidized enables a disulfide (cystine) term in ε280. Reduced assumes no disulfide contribution. Choose based on your buffer and redox conditions, or specify disulfides manually.
3) When should I use the A260 correction?
Use it when nucleic acids are present and A260 was measured for the same sample. It can reduce overestimation but is still an approximation, especially for complex mixtures.
4) My instrument reports a different path length. What do I enter?
Enter the actual path length used for the reading, in centimeters. Microvolume devices often vary path length automatically; use the value reported by the instrument for that measurement.
5) Why does dilution factor increase the final concentration?
The measurement is taken on a diluted sample. Multiplying by the dilution factor estimates the original stock concentration. If you did not dilute, keep the factor at 1.
6) How is mg/mL computed from molar concentration?
The tool multiplies molar concentration by molecular weight (g/mol) to get g/L, which equals mg/mL numerically. Provide MW in kDa for quick mass-concentration reporting.
7) What if I already know ε280 from a datasheet?
Enter it in the manual ε field. The calculator will use your value for concentration, while still showing the estimated ε for comparison and sanity checking.