Calculate extinction coefficient (ε) from a protein or DNA/RNA sequence, or solve absorbance, concentration, and path length using the Beer-Lambert law.
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Extinction Coefficient Formula
The calculator uses the protein sequence formula below, a nearest-neighbor sum for unmodified single-stranded DNA/RNA at 260 nm, or the Beer–Lambert relationship. For nucleic acids, ε equals the sum of adjacent-base-pair coefficients minus the sum of interior single-base coefficients; no universal 0.9 factor is applied.
From protein sequence (Pace/Edelhoch method, 280 nm):
- ε = molar extinction coefficient at 280 nm (M⁻¹ cm⁻¹)
- nW = number of tryptophan residues
- nY = number of tyrosine residues
- nC-C = number of cystine (disulfide) bonds; 0 if cysteines are reduced
From absorbance (Beer–Lambert law):
- A = absorbance (unitless)
- c = molar concentration (M)
- l = path length (cm)
- ε = molar extinction coefficient (M⁻¹ cm⁻¹)
The protein sequence method estimates absorbance at 280 nm in water from Trp, Tyr, and explicitly assumed disulfide bonds. Free cysteines contribute zero. It is not a measured coefficient or a guaranteed accuracy bound; proteins without Trp can have substantial errors. The default assumes reduced cysteines.
Reference Values
Per-residue contributions used in the sequence calculation:
| Group | ε at 280 nm (M⁻¹ cm⁻¹) |
|---|---|
| Tryptophan (W) | 5,500 |
| Tyrosine (Y) | 1,490 |
| Cystine (disulfide) | 125 |
| Free cysteine (-SH) | 0 |
Approximate illustrative protein values at 280 nm. Sequence, variant, solvent, modifications and measurement method matter; these entries are not calculator defaults. The insulin entry is a Pace sequence prediction.
| Protein | MW (Da) | ε (M⁻¹ cm⁻¹) | A (1 mg/mL) |
|---|---|---|---|
| BSA | 66,400 | 43,824 | 0.66 |
| IgG (typical) | 150,000 | 210,000 | 1.40 |
| Lysozyme | 14,300 | 38,940 | 2.72 |
| Human insulin (Pace prediction) | 5,808 | 6,335 | 1.09 |
| GFP (variant-dependent illustration) | 26,900 | 22,000 | 0.82 |
Worked Example
Sequence input: a 100-residue protein with 2 Trp, 5 Tyr, and 4 Cys assumed all oxidized (2 disulfides).
ε = (2 × 5500) + (5 × 1490) + (2 × 125)
ε = 11,000 + 7,450 + 250
ε = 18,700 M⁻¹ cm⁻¹
Beer–Lambert input: A₂₈₀ = 0.85, c = 0.1 mM, l = 1 cm.
ε = 0.85 / (1 × 10⁻⁴ × 1) = 8,500 M⁻¹ cm⁻¹
FAQ
Reduced or oxidized cysteines? Use the bond state established for your sample. Reduced means zero disulfides; maximum pairing is an explicit assumption, not a determination of structure. A known count must not exceed the cysteine pairs in the entered chain.
Why does my measured ε differ from the predicted value? Aggregation, scattering, bound cofactors, denaturation, or unusual local environments around aromatic residues all shift A₂₈₀. Use a validated experimental protocol appropriate for the protein and report the wavelength and solution conditions.
Can I use this for nucleic acids? Yes. Choose the unmodified single-stranded DNA or RNA nearest-neighbor mode at 260 nm. It does not model duplexes, modified bases or secondary structure.
What if my protein has no Trp or Tyr? A₂₈₀ will be very low and unreliable. Use a colorimetric assay (BCA, Bradford) or measure at 205 nm instead.
