Calculate protein molecular weight from amino acid sequence or composition. Covers residue weights, pI estimation, extinction coefficient, and charge at pH.
Protein molecular weight (MW) is one of the most fundamental properties in biochemistry. It determines migration on SDS-PAGE gels, elution volume in size exclusion chromatography, sedimentation in ultracentrifugation, and is essential for converting between mass concentration (mg/mL) and molar concentration (µM). Every protein experiment — from Western blot band identification to drug dosing — depends on knowing the molecular weight.
The molecular weight of a protein is calculated from its amino acid sequence by summing the residue weights of all amino acids and subtracting water (18.02 Da) lost at each peptide bond. The 20 standard amino acids have residue weights ranging from 57.02 Da (glycine) to 186.21 Da (tryptophan), with an average of about 110 Da. A 300-residue protein therefore has an approximate MW of ~33,000 Da (33 kDa).
This calculator computes exact MW from either a one-letter amino acid sequence or amino acid composition counts. Beyond MW, it estimates the theoretical isoelectric point (pI), molar extinction coefficient at 280 nm, and net charge at a given pH — all critical parameters derivable from sequence alone. These calculations replicate the functionality of ExPASy ProtParam, the gold-standard online tool for protein physicochemical properties.
Protein MW calculation is essential for SDS-PAGE gel interpretation, mass spectrometry data validation, buffer preparation, and converting between mass and molar concentrations. This calculator provides instant, accurate results from sequence input. This protein molecular weight calculator helps you compare outcomes quickly and reduce avoidable mistakes when making day-to-day care decisions. Use the estimate as a planning baseline and confirm final decisions with a qualified professional when risk is high.
MW = Σ(residue masses) + 18.02 (water for termini). Residue mass = amino acid MW - 18.02 (water lost per peptide bond). Extinction coefficient: ε₂₈₀ = nTrp × 5500 + nTyr × 1490 + nCys-Cys × 125 (M⁻¹cm⁻¹). Theoretical pI: calculated by iterating pH until net charge = 0.
Result: MW = 26,899 Da (26.9 kDa)
GFP (238 amino acids): sum of all residue masses + terminal water = 26,899 Da. Contains 1 Trp, 11 Tyr, 2 Cys → ε₂₈₀ = 21,890 M⁻¹cm⁻¹.
The 20 standard amino acids have these residue masses (monoisotopic): **Gly (G)** 57.02, **Ala (A)** 71.04, **Val (V)** 99.07, **Leu (L)** 113.08, **Ile (I)** 113.08, **Pro (P)** 97.05, **Phe (F)** 147.07, **Trp (W)** 186.08, **Met (M)** 131.04, **Ser (S)** 87.03, **Thr (T)** 101.05, **Cys (C)** 103.01, **Tyr (Y)** 163.06, **His (H)** 137.06, **Asp (D)** 115.03, **Glu (E)** 129.04, **Asn (N)** 114.04, **Gln (Q)** 128.06, **Lys (K)** 128.09, **Arg (R)** 156.10. These are average masses; monoisotopic masses (using most abundant isotope) differ slightly and are used in high-resolution mass spectrometry.
The theoretical pI is computed by iterating pH from 0 to 14 and calculating the net charge at each pH using the Henderson-Hasselbalch equation for each ionizable group. **pKa values used**: N-terminus = 9.69, C-terminus = 2.34, Asp (D) = 3.65, Glu (E) = 4.25, Cys (C) = 8.18, Tyr (Y) = 10.07, His (H) = 6.00, Lys (K) = 10.54, Arg (R) = 12.48. The pH where net charge crosses zero is the pI. Multiple pI calculators exist (ExPASy, IPC, Bjellqvist) and may give slightly different results depending on the pKa values used.
Common unstained MW markers: **10 kDa** (Aprotinin), **15 kDa** (Lysozyme), **25 kDa**, **35 kDa**, **40 kDa**, **55 kDa**, **70 kDa** (BSA), **100 kDa**, **130 kDa**, **170 kDa**, **250 kDa**. For accurate MW estimation by gel migration, plot log(MW) vs relative mobility (Rf) for the standards and interpolate your unknown. The linear range depends on gel percentage: 15% for 10-60 kDa, 10% for 15-150 kDa, 7.5% for 30-300 kDa.
SDS-PAGE estimates MW based on the assumption that all proteins bind SDS uniformly (1.4 g SDS per g protein). Aberrant migration occurs with: glycosylated proteins (run higher), highly charged proteins, proline-rich proteins, membrane proteins (bind more SDS), and very basic proteins. The predicted sequence MW is always correct; the gel migration is the approximation.
It depends on the mature form you're working with. If your protein is secreted and the signal peptide is cleaved, calculate MW without it. If you're expressing in E. coli without signal peptide processing, include it. Add the His-tag (6× His = 840 Da) or other fusion tags to the total.
The Pace method (based on Trp/Tyr/Cys content) is accurate to within 5-10% for most native proteins. Assumptions: all Cys form disulfide bonds (if unknown, calculate both with and without Cys contribution). Denatured proteins have slightly different ε₂₈₀ values. For maximum accuracy, measure experimentally by amino acid analysis.
The pI is the pH where the protein has zero net charge. Below pI, the protein is positively charged; above pI, negatively charged. The pI determines behavior in ion exchange chromatography and isoelectric focusing. Computed pI assumes the protein is unfolded — actual pI may differ due to buried residues and post-translational modifications.
Common PTMs: phosphorylation (+80 Da per site), glycosylation (+162 per hexose), acetylation (+42), ubiquitination (+8,565 per Ub), methionine oxidation (+16), disulfide bond formation (-2 Da per bond). For mass spectrometry, these shifts identify the modification type and site.
This calculator covers the 20 standard amino acids. For selenocysteine (Sec, U, 150.04 Da) or pyrrolysine (Pyl, O, 255.31 Da), substitute the appropriate mass. For synthetic peptides with D-amino acids, the masses are identical to L-forms.