Protein Molar Extinction Coefficient Calculator
Predict a protein’s ε at 280 nm from a pasted amino-acid sequence or from its tryptophan, tyrosine and cystine counts (Pace / ProtParam or Gill & von Hippel), with molecular weight, Abs 0.1%, oxidised vs reduced values and the concentration from an A280 reading.
How it works
ε₂₈₀ = 5,500·nTrp + 1,490·nTyr + 125·nCystine (M⁻¹cm⁻¹, Pace 1995; Gill & von Hippel: 5,690 / 1,280 / 120) · MW = Σ residue masses + 18.02 · A280 (1 mg/mL) = ε ÷ MW · c = A ÷ (ε·l) × dilution
At 280 nm a protein’s absorbance comes almost entirely from tryptophan, tyrosine and disulfide-bonded cysteine (cystine), so counting those residues predicts the extinction coefficient within a few percent. Paste the sequence and the calculator counts them and works out the average molecular weight from the residue masses, the same way ExPASy ProtParam does — or type the counts and the weight yourself. Like ProtParam it reports two values when there are cysteines: one assuming every pair forms a disulfide (for a folded, oxidised protein) and one assuming all are reduced (in DTT or TCEP); you can also enter the exact number of disulfide bonds. Dividing ε by the molecular weight gives the absorbance of a 1 mg/mL solution (ε 0.1%), the number most spectrophotometers ask for; E1% is ten times that. The default coefficients are those of Pace et al. (1995); the older Gill & von Hippel set is offered for comparison. Free cysteines contribute nothing, and nucleic-acid contamination or light scattering inflate readings.
Worked example
BSA by counts: 2 Trp, 20 Tyr, 17 disulfides → ε = 11,000 + 29,800 + 2,125 = 42,925 M⁻¹cm⁻¹ (the accepted value is 43,824); with MW 66,430 a 1 mg/mL solution reads 0.646. Hen egg-white lysozyme by sequence: 129 residues with 6 Trp, 3 Tyr and 8 Cys → 33,000 + 4,470 + 4 × 125 = 37,970 M⁻¹cm⁻¹ oxidised (37,470 reduced), MW 14,313.14 Da and Abs 0.1% = 2.653 — the same as ProtParam.
Sources: ExPASy ProtParam documentation · Pace CN et al. (1995) How to measure and predict the molar absorption coefficient of a protein. Protein Science 4:2411–2423 · Gill SC, von Hippel PH (1989) Calculation of protein extinction coefficients from amino acid sequence data. Anal Biochem 182:319–326
Frequently asked questions
Reduced or oxidised?
Use the oxidised value (all cysteines paired) for a folded protein that has disulfides, the reduced value in buffers with DTT, TCEP or β-mercaptoethanol, and the custom option if you know how many disulfide bonds the structure has. The difference is only 125 M⁻¹cm⁻¹ per bond.
Does the buffer matter?
Blank against the exact buffer — Tris, imidazole, detergents and nucleotides absorb near 280 nm. Guanidine-denatured proteins give values close to the predicted ε, which is how the coefficients were calibrated.
What if my protein has no tryptophan?
The coefficient then depends only on tyrosines and cystines and is small, so the relative error grows to 10% or more. Use a dye assay (Bradford, BCA) or measure ε experimentally.
Should I include the signal peptide or tags?
Paste the sequence of the protein as it is in the tube: without a cleaved signal peptide, but with any His-tag or fusion partner that is still attached, since a tag’s tryptophans and tyrosines absorb too.
How do I go from molar concentration to mg/mL?
Multiply by the molecular weight: 15.1 µM of a 66,430 Da protein is 15.1 × 10⁻⁶ × 66,430 ≈ 1.0 mg/mL. The calculator shows both.
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