Estimate protein concentration from an A280 absorbance reading. Enter the A280, your dilution and path length, and the protein’s extinction coefficient (or pick a preset).
Concentration (mg/mL) = A280 × dilution / (E0.1% × path). E0.1% is the A280 of a 1 mg/mL solution — it is protein-specific (generic ≈1.0, IgG ≈1.4, BSA ≈0.66). For accuracy, compute it from the protein's sequence.
How the A280 method works
Aromatic residues (mainly tryptophan and tyrosine) absorb UV at 280 nm, so absorbance is proportional to protein concentration:
concentration (mg/mL) = A280 × dilution / (E0.1% × path length)
where E0.1% (the 0.1% extinction coefficient) is the A280 of a 1 mg/mL solution in a 1 cm cuvette. It is protein-specific because it depends on the Trp/Tyr/Cys content.
Choosing the right coefficient
- Generic (≈1.0): the common “1 A280 ≈ 1 mg/mL” assumption — convenient but only approximate.
- IgG (≈1.4): antibodies.
- BSA (≈0.66): a frequent standard.
- Your protein: for accuracy, calculate E from the sequence (e.g. with ExPASy ProtParam) and enter it.
Watch for interference
- Nucleic acid contamination absorbs at 280 nm and inflates the reading — check the A260/A280 ratio (≈0.57 for pure protein).
- Light scattering from aggregates raises baseline absorbance; a turbid sample needs clarifying.
- For proteins with few aromatic residues, A280 is unreliable — use a colorimetric assay (Bradford, BCA).
Frequently asked questions
Does 1 A280 equal 1 mg/mL?
Only as a rough generic estimate. The true factor depends on the protein’s extinction coefficient (e.g. IgG ≈1.4, BSA ≈0.66).
How do I get my protein’s extinction coefficient?
Calculate it from the amino acid sequence with a tool like ExPASy ProtParam, then enter it as E0.1%.
When should I not use A280?
When the protein has few Trp/Tyr residues, or the sample has nucleic acid contamination or scattering — use Bradford or BCA instead.
Related tool: Protein MW & isoelectric point →
