Protein concentration from A280

Proteins absorb ultraviolet light at 280 nm, mostly through their aromatic side chains. Measure that absorbance and, if you know the protein’s sequence, you can convert it to a concentration in mg/mL — properly, not with a blanket assumption.

Beer–Lambert

Absorbance is proportional to concentration through the molar extinction coefficient ε and the path length:

molar concentration = A280 ÷ (ε · path length)

Multiply the molar concentration by the protein’s molecular weight to get mg/mL. The path length is usually 1 cm (or handled for you on a NanoDrop).

ε comes from the sequence

Absorbance at 280 nm is dominated by two residues:tryptophan (5,500) and tyrosine (1,490)M⁻¹cm⁻¹ each, with a small contribution from disulfide-bonded cystines (125 per pair). Counting those residues gives ε directly — so a protein with one Trp and one Tyr has ε = 6,990 M⁻¹cm⁻¹.

A worked example

A protein with ε = 6,990 M⁻¹cm⁻¹ and MW 13,970 Da reading A280 = 1.0: molar = 1.0 ÷ 6,990 = 1.43×10⁻⁴ M, so mg/mL = 1.43×10⁻⁴ × 13,970 ≈2.0 mg/mL. Because ε is derived from the actual aromatic content, this beats the rough “1 A280 = 1 mg/mL” rule, which is only ever right by coincidence.

The catch: no Trp or Tyr, no signal

A protein with neither tryptophan nor tyrosine barely absorbs at 280 nm, so A280 can’t quantify it — ε would be near zero. Those proteins need a colorimetric assay (BCA, Bradford) instead. Nucleic-acid contamination also inflates A280 via its own 280 nm absorbance, so check the A260/A280 ratio alongside.