Protein Molar Extinction Coefficient Calculator

Molar Extinction Coefficient (ε) The molar extinction coefficient ε (also called molar absorptivity) quantifies how strongly a protein absorbs light at 280 nm. Beer-Lambert law: A = ε × l × c Where A = absorbance, l = path length (cm), c = molar concentration (M). At 280 nm, absorption is dominated by aromatic amino acids.

Pace Formula (1995) ε₂₈₀ = (nTrp × 5,500) + (nTyr × 1,490) + (nCys-Cys × 125) Where: nTrp = number of tryptophan residues (absorbs most strongly) nTyr = number of tyrosine residues nCys-Cys = number of disulfide bonds (cystine) — NOT free cysteine

Units: M⁻¹cm⁻¹ (also written as L·mol⁻¹·cm⁻¹) Source: Pace et al., Protein Science, 1995.

Predicting Protein Concentration From absorbance at 280 nm: c (mg/mL) = (A₂₈₀ × MW) / (ε₂₈₀ × path length) c (μM) = (A₂₈₀ × 10⁶) / (ε₂₈₀ × path length)

Why 280 nm? Trp and Tyr absorb UV light near 280 nm due to their aromatic rings. This is convenient because buffers, salts, and most contaminants don’t absorb at 280 nm. DNA/RNA absorb strongly at 260 nm — contamination shifts A280 up artificially. Phenylalanine (Phe) has very weak absorption at 280 nm; usually ignored.

Typical Values Small proteins (no Trp, 0 Tyr): ε ≈ 0 (cannot use A280) Typical enzyme (2–5 Trp): ε ≈ 10,000–30,000 M⁻¹cm⁻¹ Antibodies: ε ≈ 200,000–220,000 M⁻¹cm⁻¹ BSA (bovine serum albumin): ε ≈ 43,824 M⁻¹cm⁻¹ Lysozyme: ε ≈ 36,000 M⁻¹cm⁻¹