Peptide Extinction Coefficient Calculator
The molar extinction coefficient at 280 nm (ε280, M⁻¹ cm⁻¹) describes how strongly a peptide absorbs light at 280 nm. With a measured A280, path length and dilution factor, it lets you estimate concentration.
Results
From sequence to measurement
Calculate ε280 first. Add an absorbance reading to see the concentration of the measured sample and the original solution.
| Component | Count | M⁻¹ cm⁻¹ | Share |
|---|---|---|---|
| Tryptophan (W) | |||
| Tyrosine (Y) | |||
| Disulfide bonds |
Total cysteines in sequence: .
Add a sample A280 to calculate concentration.
Concentration
- Corrected A280
- Sample − blank
- Measured sample
- Path:
- Original solution
- Dilution:
- Original mass concentration
Worked example: WYCC
- Sample A280
- 0.37575
- Blank A280
- 0.02
- Corrected A280
- 0.35575
- Path used in the formula
- 1 cm
- Original solution before 2× dilution
- 100 µM
Illustrative synthetic example, not laboratory data: the sequence WYCC with one documented intrachain disulfide bond gives ε280 = 5500 + 1490 + 125 = 7115 M⁻¹ cm⁻¹. At a 1 cm path, the corrected A280 is 0.37575 − 0.02000 = 0.35575. Dividing by 7115 M⁻¹ cm⁻¹ × 1 cm gives a measured 50 µM, and a 2× dilution factor gives an original 100 µM. The same sample read raw at 1 mm would show 0.037575 with a 0.002 blank. Entering 1 mm as the path gives the same 50 µM measured result. A blank-corrected value normalized to 10 mm would read 0.35575 and should be entered with the fixed 1 cm path.
How the A280 calculation works
- Correct the reading Sample A280 − matched blank
- Find molar concentration Corrected A280 ÷ (ε280 × path in cm)
- Undo dilution Measured concentration × dilution factor
Estimate ε280 from sequence
Sequence estimate: Enter the single peptide chain you actually measured, as 1 to 500 standard one-letter residues in plain text or one FASTA record. The calculator counts tryptophan (W), tyrosine (Y) and cysteine (C) and applies the residue model described by Pace et al. (1995) and the ExPASy ProtParam documentation: 5500 M⁻¹ cm⁻¹ per W, 1490 per Y and 125 per documented intrachain disulfide bond. If the sequence has two or more cysteines, enter the documented number of intrachain disulfide bonds. Zero is valid, and the maximum is half the cysteine count, rounded down. The calculator never infers pairing. For modified or nonstandard residues, or for unknown or complex bonding, switch to Known ε280 mode and enter an applicable coefficient.
Convert A280 to concentration
Reading to concentration: Corrected A280 = sample A280 − blank A280. Measured concentration (M) = corrected A280 ÷ (ε280 × path in cm). Original concentration = measured concentration × dilution factor. Match the path to how your instrument reports the reading. For raw values, enter the actual optical path in cm or mm. For values normalized to a 10 mm path, use the fixed 1 cm option. Subtract a matched blank only if the reading is not already blank-corrected, and only once, on the same basis as the sample. If you enter an applicable molecular weight in g/mol, the original concentration is also shown in mg/mL (M × g/mol).
Know the limits
Limitations: Sequence-derived coefficients are estimates. The actual chemical form of the peptide, its modifications and its bonding can change the true value. If the sequence has no W or Y, the calculator still shows the estimate, including any cystine contribution, but turns off concentration in sequence mode. Absorbance at 280 nm may be weak for these peptides. Use an applicable measured coefficient or a validated alternative method instead. Estimates for sequences that contain Y but no W carry more uncertainty. Check your instrument's documentation for its reliable absorbance range. Results are for research calculations only.
Peptide extinction coefficient and A280 FAQs
How do I calculate peptide concentration from A280?
Subtract any matched blank to get the corrected A280. Divide it by ε280 (M⁻¹ cm⁻¹) times the path length in cm to get the measured molar concentration. Then multiply by the dilution factor to get the original concentration.
Can I use A280 for a peptide without tryptophan or tyrosine?
Not reliably with this calculator. These peptides may give only a weak signal at 280 nm, and a sequence-only cystine estimate does not justify a concentration result. Use an applicable measured coefficient in Known ε280 mode, or a validated alternative method.
Why do calculators give different extinction coefficients?
Calculators use different model constants and handle cysteine differently, and the actual chemical form of the peptide also matters. This calculator uses 5500 M⁻¹ cm⁻¹ per W, 1490 per Y and 125 per documented intrachain disulfide bond, and it does not assume pairing. Every sequence-derived value is an estimate.
Which peptide sequence should I enter?
Enter the single chain you actually measured, in standard one-letter format, as plain text or one FASTA record. If the peptide has modifications, nonstandard residues, or unknown or complex bonding, use an applicable known coefficient instead.
Do I use the instrument path or 1 cm for A280?
Use the path that matches how the reading is reported. For raw values, enter the actual optical path. For values normalized to a 10 mm path, use 1 cm. Check your instrument's documentation, because no single normalized path applies to every instrument.
Should I subtract the blank before calculating concentration?
Yes, if the reading is not already blank-corrected. Use a blank in a matched matrix, reported on the same basis as the sample, and subtract it only once.
Method references
- Pace et al. (1995), protein molar absorption coefficient at 280 nm
- ExPASy ProtParam method documentation
- Anthis and Clore (2013), quantification using absorbance at 205 nm
- Thermo Fisher, measuring protein concentration at 280 nm
- NanoDrop One user guide
