Extinction Coefficient Calculator

How to Use the Extinction Coefficient Calculator

1. Paste your protein amino acid sequence in single-letter code, or upload a .txt / .fasta file.
2. Select the cysteine condition — choose Reduced if no disulfide bonds are present, or Oxidized if disulfide bonds exist (e.g. native secreted proteins).
3. Optionally enter the molecular weight (Da) to get the specific absorbance E1% (A280 at 1 mg/mL, 1 cm pathlength).
4. Click Calculate Extinction Coefficient.
5. Use the Beer-Lambert Concentration Calculator at the bottom of the result to convert any A280 reading to protein concentration (µM and mg/mL).

About the Extinction Coefficient at 280 nm

Proteins absorb ultraviolet light at 280 nm primarily due to three aromatic and sulfur-containing residues: Tryptophan (Trp / W), Tyrosine (Tyr / Y), and Cysteine (Cys / C) when involved in disulfide bonds.

The molar extinction coefficient (ε, epsilon) is calculated using the Pace et al. (1995) formula, which is the standard method used by ExPASy ProtParam:

ε = (nW × 5500) + (nY × 1490) + (nSS × 125)

Where nW = number of Trp, nY = number of Tyr, nSS = number of disulfide bonds (= nCys / 2 if oxidized). The values 5500, 1490, and 125 M⁻¹cm⁻¹ are the individual residue extinction coefficients at 280 nm.

Once ε is known, protein concentration is calculated from the Beer-Lambert law: C = A / (ε × l), where A is the absorbance and l is the pathlength in cm.

Reduced vs Oxidized Cysteine

• Reduced (free thiols): Free cysteine residues do not significantly absorb at 280 nm. Use this for denatured proteins or intracellular proteins with no disulfide bonds.
• Oxidized (disulfide bonds): Each disulfide bond adds 125 M⁻¹cm⁻¹ to the extinction coefficient. Use this for native secreted proteins, antibodies, or any protein known to contain disulfide bonds.
• If unsure, calculate both and compare — the difference is usually small unless the protein has many Cys residues and no Trp.