Protein molecular weight, pI and extinction coefficient calculator
Paste a protein sequence in one-letter code or FASTA and the calculator adds up the residue masses to give the molecular weight (average and monoisotopic), predicts the molar extinction coefficient at 280 nm from its Trp, Tyr and cystine content (ε280 = 5500 × Trp + 1490 × Tyr + 125 × cystine, Pace et al. 1995), and finds the theoretical isoelectric point and net charge from published pK values. Enter an A280 reading to get the protein concentration in mg/mL and µM.
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At a glance
| Input | Protein sequence in one-letter code or FASTA (20 standard amino acids, Sec and Pyl) |
|---|---|
| Molecular weight | Sum of residue masses + H₂O; CIAAW 2021 atomic weights (average) and AME 2020 isotope masses (monoisotopic) |
| ε280 | 5500 × Trp + 1490 × Tyr + 125 × cystine (Pace et al. 1995) |
| pI | Henderson–Hasselbalch, pK values in folded proteins (Grimsley et al. 2009; Arg 13.8, Fitch et al. 2015) |
| Output | MW, monoisotopic mass, formula, pI, net charge, ε280, Abs 0.1%, composition, A280 concentration; CSV |
| Runs in | Your web browser; nothing is uploaded |
| Price | Free, no account needed |
What it does
How to use the protein parameter calculator
- Paste the sequencePaste the protein sequence in one-letter code, or a FASTA record. Spaces, line breaks and position numbers are ignored; only the first FASTA record is used.
- Check what was readThe length and elemental formula are shown with the result; letters that are not amino acids are listed with their positions.
- Read the parametersRead the molecular weight, monoisotopic mass, pI, ε280 and composition. Change the pH to see the net charge at your buffer pH.
- Get the concentrationEnter a blank-corrected A280 and the path length, choose ε with cystines (disulfide bonds) or reduced Cys, and read the concentration in mg/mL and µM.
- ExportCopy the summary, download the CSV, or send the molecular weight to the molarity calculator.
How it works
Molecular weight. Each residue is its amino acid less one water; the protein is the sum of its residues plus one water for the free N- and C-termini:
M = Σ nᵢ × Mresidue(i) + M(H₂O)
Residue masses are computed from their elemental formulas. The average mass uses the IUPAC (CIAAW) 2021 standard atomic weights (Prohaska et al. 2022), the same values as the molar mass calculator; the monoisotopic mass uses the mass of the most abundant isotope of each element (¹H, ¹²C, ¹⁴N, ¹⁶O, ³²S) from the AME 2020 atomic mass evaluation (Wang et al. 2021). Every cysteine is counted as reduced (–SH); each disulfide bond removes two hydrogens, 2.016 Da.
Extinction coefficient. Pace et al. (1995) fitted 116 measured values for 80 proteins and found the molar absorption coefficient at 280 nm of a folded protein in water is best predicted by
ε280 (M⁻¹ cm⁻¹) = 5500 × n(Trp) + 1490 × n(Tyr) + 125 × n(cystine)
The calculator gives it twice: with every pair of Cys as a cystine (disulfide), and with every Cys reduced (no cystine term). The prediction is reliable for proteins with Trp and less so for those without, and Pace et al. recommend measuring ε when it matters (Pace et al. 1995). Abs 0.1% is the absorbance of a 1 g/L solution in a 1 cm cell, ε / M.
Concentration from A280 follows the Beer–Lambert law: c (M) = A280 / (ε × l), and c (mg/mL) = c (M) × M.
Isoelectric point. The net charge at a pH is the sum, from the Henderson–Hasselbalch equation, of the positive charge on the N-terminus, Lys, Arg and His and the negative charge on the C-terminus, Asp, Glu, Cys and Tyr; the pI is the pH where it is zero, found by bisection (Kozlowski 2016). The pK values are the averages measured in folded proteins by Grimsley et al. (2009): N-terminus 7.7, C-terminus 3.3, Asp 3.5, Glu 4.2, His 6.6, Cys 6.8, Tyr 10.3 and Lys 10.5; that study gives none for arginine, so Arg uses 13.8 (Fitch et al. 2015). Other tools use other pK sets (Kozlowski compared 15), so their pI can differ by a few tenths; modifications such as phosphorylation are not included.
Worked example
Angiotensin II, DRVYIHPF (sequence as given by Santos et al. 2008). The residues add up to C₅₀H₇₁N₁₃O₁₂: 50 × 12.011 + 71 × 1.0080 + 13 × 14.007 + 12 × 15.999 = 1046.20 Da average; with ¹²C, ¹H, ¹⁴N and ¹⁶O the monoisotopic mass is 1045.5345 Da.
It has one Tyr, no Trp and no Cys, so ε280 = 1 × 1490 = 1490 M⁻¹ cm⁻¹ and Abs 0.1% = 1490 / 1046.2 = 1.424. A blank-corrected A280 of 0.85 in a 1 cm cell gives 0.85 / 1490 = 570.5 µM, or 570.5 µM × 1046.2 g/mol = 0.597 mg/mL. Without Trp the predicted ε is less reliable, as Pace et al. note, so the calculator says so.
With Asp, Arg, His, Tyr and the two termini, the charge is zero at a theoretical pI of 7.15, and the net charge at pH 7.4 is −0.20.
Frequently asked questions
How is the molecular weight of a protein calculated from its sequence?
Each amino acid loses one water when it joins the chain, so the molecular weight is the sum of the residue masses plus one water (18.015 Da) for the two ends. The calculator does this from each residue’s elemental formula and the CIAAW 2021 atomic weights, with every cysteine reduced.
What is the difference between average and monoisotopic mass?
The average mass uses atomic weights averaged over the natural isotopes and is what you use for weighing and molarity. The monoisotopic mass uses only the most abundant isotope of each element (¹²C, ¹H, ¹⁴N, ¹⁶O, ³²S) and matches the lightest peak in a mass spectrum of a peptide.
How do I calculate the extinction coefficient of a protein?
Count the Trp, Tyr and cystines and use ε280 = 5500 × Trp + 1490 × Tyr + 125 × cystine, in M⁻¹ cm⁻¹ (Pace et al. 1995). Use the cystine value if the protein has disulfide bonds and the reduced value (no cystine term) if it is reduced or has no disulfides.
How do I get protein concentration from A280?
Divide the blank-corrected absorbance by ε280 times the path length to get the molar concentration (A = ε c l), then multiply by the molecular weight for mg/mL. Equivalently, mg/mL = A280 / Abs 0.1% for a 1 cm path.
Why does my pI differ from ExPASy ProtParam or other tools?
The theoretical pI depends on the pK values used, and tools use different sets. This calculator uses pK values measured in folded proteins (Grimsley et al. 2009, with Arg 13.8 from Fitch et al. 2015); ProtParam and others use other sets, so the values can differ by a few tenths of a pH unit. Phosphorylation and other modifications are not included.
Why is the predicted extinction coefficient flagged for proteins without tryptophan?
Pace et al. (1995) found the predicted ε280 is quite reliable for proteins that contain Trp and less reliable for those that do not, and recommend measuring ε in that case.
Can I paste a FASTA file or a DNA sequence?
You can paste a FASTA record; the title line is kept and only the first record is used. DNA or RNA has to be translated into protein first; a sequence of only A, C, G, T and U is flagged.
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