Protein Concentration from A280

Paste the amino-acid sequence of your protein for its molar extinction coefficient at 280 nm (from its tryptophans, tyrosines and disulfide bonds) and its molecular weight. Add an absorbance reading for the concentration in mg/mL and µM, without a standard curve.

BiochemistrySpectrophotometryProteins

Inputs

Input examples

Use the one-letter code of the mature protein (without a signal peptide that is cut off); spaces, numbers and a FASTA header line are ignored. Only tryptophan (W), tyrosine (Y) and disulfide-bonded cysteines (cystines) absorb noticeably at 280 nm. Choose All cysteines reduced if you measure in a reducing buffer (DTT, β-mercaptoethanol) or the protein has no disulfide bonds. A protein without W or Y cannot be measured at 280 nm; use A205 or a colour assay (BCA, Bradford) instead.

Protein sequence

Cysteines

A280 — optional

Dilution factor

Light path — mm

Why 280 nm Works for Proteins

The aromatic side chains of tryptophan and tyrosine absorb ultraviolet light around 280 nm; disulfide bonds add a little. Pace and co-workers showed in 1995 that adding up their contributions predicts the extinction coefficient of a folded protein to within a few percent, so a single absorbance reading gives the concentration without a standard curve.

Because the result depends on how many W and Y a protein has, “1 absorbance unit = 1 mg/mL” is only a rough average: the value for 1 mg/mL (the Abs 0.1%) ranges from almost 0 for proteins without tryptophan to above 3 for tryptophan-rich ones. Nucleic acids also absorb at 280 nm, so a high A260/A280 means DNA or RNA contamination.

W5500 L/(mol·cm) each

Y1490 each

cystine125 per S–S bond

A260/A280 > 0.6nucleic acid contamination

Amino-Acid Composition

residues

W, Y and C: absorb at 280 nm

other residues

Result

Paste a sequence above

Worked Techniques

Count the Absorbers

Hen egg-white lysozyme has 6 W, 3 Y and 8 cysteines forming 4 disulfide bonds.

Example: reduced, the value drops to 37 470 L/(mol·cm).

Beer–Lambert

Divide the absorbance by ε and the light path in cm for mol/L; multiply by the molecular weight for g/L.

Example: 13.2 µM × 14 313 g/mol = 0.188 mg/mL.

Molecular Weight

Add the average mass of every residue in the chain plus one water for the free ends (average isotopic masses, as in ExPASy ProtParam).

Example: ubiquitin, 76 residues: about 8565 g/mol, with no tryptophan, so ε₂₈₀ = 1490 only.

Sources & Verification

References

C. N. Pace, F. Vajdos, L. Fee, G. Grimsley and T. Gray, “How to measure and predict the molar absorption coefficient of a protein”, Protein Science 4 (1995) 2411–2423 — ε280 = 5500 W + 1490 Y + 125 cystines.
E. Gasteiger et al., “Protein identification and analysis tools on the ExPASy server”, in The Proteomics Protocols Handbook, Humana Press, 2005 — ProtParam: average residue masses and the extinction coefficient with all cysteines as cystines or reduced.