Calculate protein concentration from UV absorbance at 280 nm.
Choose your mode depending on what extinction coefficient you have. "Molar ε" uses the standard Beer-Lambert approach (units of M⁻¹ cm⁻¹). "Mass ε" uses the ε₀.₁% convention (also called A₀.₁% or the absorbance of a 1 mg/mL solution), which gives concentration directly in mg/mL without needing the molecular weight.
If you measured on a NanoDrop or similar microvolume spectrophotometer, the path length is typically 1 mm (0.1 cm) — make sure to adjust accordingly.
UV absorbance at 280 nm measures the aromatic amino acids tryptophan (Trp, ε₂₈₀ ≈ 5,500 M⁻¹cm⁻¹), tyrosine (Tyr, ε₂₈₀ ≈ 1,490 M⁻¹cm⁻¹), and to a lesser extent cystine (disulfide bonds, ε₂₈₀ ≈ 125 M⁻¹cm⁻¹). The molar extinction coefficient of a protein can be estimated from its amino acid composition using the Pace equation: ε₂₈₀ = (nTrp × 5500) + (nTyr × 1490) + (nCys-Cys × 125).
Using molar ε: c (M) = A₂₈₀ / (ε × l), then convert to mg/mL by multiplying by MW/1000.
Using mass ε₀.₁%: c (mg/mL) = A₂₈₀ / (ε₀.₁% × l). This is convenient because you get mg/mL directly.
Check the A₂₈₀/A₂₆₀ ratio to assess nucleic acid contamination. Pure protein typically gives a ratio of ~1.8. Ratios below 0.6 suggest significant nucleic acid contamination, which will inflate your protein concentration estimate.