Protein Molecular Weight, Isoelectric Point & 280 nm Extinction Coefficient Calculator

Three quantities, three sets of references — all sources cited explicitly:

Molecular weight: Sum of the average residue masses of each amino acid, plus one water molecule (18.015). Uses the ExPASy average mass table, not monoisotopic masses — the monoisotopic peak seen by mass spectrometry will be slightly lower than this value.

Theoretical isoelectric point pI: Uses pKa values from EMBOSS iep, solved by bisection for the pH at which net charge = 0. pKa values used: N-terminus 8.6, C-terminus 3.6, Asp 3.9, Glu 4.1, Cys 8.5, His 6.5, Tyr 10.1, Lys 10.8, Arg 12.5 (Source: EMBOSS iep documentation).

Molar extinction coefficient ε₂₈₀, using the empirical formula from Pace et al. 1995:

ε₂₈₀ (M⁻¹cm⁻¹) = 5500 × nTrp + 1490 × nTyr + 125 × n_SS

Source: Pace, Vajdos, Lee, Grimsley & Gray, Protein Science 4:2411–2423 (1995); the same values are used by ProtParam and EMBOSS.

Known limitations:

• Theoretical pI assumes no electrostatic interactions and no post-translational modifications. Measured pI often differs from the theoretical value by more than 0.5; phosphorylation and glycosylation shift the measured value lower. Use this to narrow down isoelectric focusing conditions, not as a precise absolute value.
• ε₂₈₀ accounts only for Trp, Tyr, and disulfide bonds. A protein containing neither Trp nor Tyr cannot be quantified by A280; use BCA or Bradford instead.
• “A280 at 1 mg/mL” = ε₂₈₀ / molecular weight, used to convert absorbance directly to mg/mL.