Protein Concentration from A280 Calculator
Convert an absorbance at 280 nm into protein concentration (mg/mL and µM) with ε (0.1%), E1% or molar ε + molecular weight, or BSA, IgG and lysozyme presets — with dilution, path length and baseline corrections, A260/A280 purity check and Warburg–Christian estimate.
How it works
c (mg/mL) = (A280 − baseline) ÷ (ε₀.₁% × path) × dilution · ε₀.₁% = E1% ÷ 10 = molar ε ÷ MW · c (M) = c (g/L) ÷ MW · Warburg–Christian: c ≈ 1.55·A280 − 0.76·A260
Tryptophan, tyrosine and cystine absorb at 280 nm, so a protein’s absorbance scales with how many of each it contains — that is its extinction coefficient. Instruments and papers quote it three ways, and the calculator takes any of them: ε (0.1%) is the absorbance of a 1 mg/mL solution, E1% (the NanoDrop setting) is the absorbance of a 10 mg/mL solution, and the molar ε in M⁻¹cm⁻¹ from ProtParam needs the molecular weight. BSA, IgG and lysozyme presets use the standard values. The generic “1 A280 = 1 mg/mL” rule can be off by 50% for proteins rich or poor in aromatics, as the comparison table shows. Readings are most reliable between about 0.1 and 1.5; above that, dilute and enter the dilution factor. A baseline reading at 320 or 340 nm can be subtracted to remove scattering from aggregates, and an optional A260 gives the 260/280 ratio (about 0.57 for pure protein) and the Warburg–Christian estimate, which roughly corrects for nucleic-acid contamination.
Worked example
BSA: A280 = 0.65 in a 1 cm cuvette, undiluted. With ε (0.1%) = 0.667, c = 0.65 ÷ 0.667 = 0.975 mg/mL, or 0.975 ÷ 66,430 = 14.7 µM. The generic value of 1.0 would give 0.650 mg/mL — a third too low. With A260 = 0.40 the ratio is 0.62 and the Warburg–Christian estimate is 1.55 × 0.65 − 0.76 × 0.40 = 0.70 mg/mL.
Sources: Thermo Fisher — Spectrophotometric determination of protein concentration at 280 nm (technical note) · ExPASy ProtParam documentation (extinction coefficients)
Frequently asked questions
How do I convert molar ε to ε (0.1%) or E1%?
ε (0.1%) = molar ε ÷ molecular weight, and E1% is ten times that. For BSA: 43,824 ÷ 66,463 = 0.66, so E1% ≈ 6.6. You can also pick the molar option and let the calculator convert.
NanoDrop path length?
The NanoDrop measures at 1.0 or 0.2 mm (or auto-ranges) but reports absorbance normalised to a 10 mm path, so use 1 cm here. For a plate reader, enter the actual path (often 0.2–0.6 cm for 100–200 µL in a 96-well plate).
Why does the 1 mg/mL = 1.0 rule not always work?
Each protein has a different number of tryptophans and tyrosines. IgG reads about 1.37 per mg/mL and BSA only 0.667, so the generic value can be far off — the table under the results shows how much.
What absorbance range is reliable?
Roughly 0.1 to 1.5 in a 1 cm cuvette. Below 0.1 noise and the blank dominate; above 1.5 stray light makes the reading non-linear. Dilute high samples and enter the dilution factor.
What does the A260/A280 ratio tell me?
Pure protein gives about 0.57; nucleic acid absorbs about twice as strongly at 260 as at 280, so a higher ratio means DNA or RNA is inflating A280. The Warburg–Christian formula (1.55·A280 − 0.76·A260) is a rough correction.
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For teaching and planning — verify against your lab protocol and instrument documentation.