protein molar extinction coefficient calculator

What is a protein molar extinction coefficient?

The molar extinction coefficient (often written as ε) tells you how strongly a protein absorbs UV light at a specific wavelength, typically 280 nm. In practice, this value lets you convert UV absorbance into concentration using Beer–Lambert law: A = εcl, where A is absorbance, c is concentration, and l is path length.

For proteins, absorbance at 280 nm is dominated by aromatic residues: tryptophan (W), tyrosine (Y), and to a smaller extent disulfide-linked cysteines (cystine). This makes sequence-based prediction a useful first estimate when preparing standards, quantifying purified proteins, or planning spectroscopy experiments.

Formula used by this calculator

This page applies the standard approximation:

ε₂₈₀ (M^-1cm^-1) = 5500 × n(W) + 1490 × n(Y) + 125 × n(disulfide bonds)

• n(W) = number of tryptophan residues
• n(Y) = number of tyrosine residues
• n(disulfide bonds) = number of Cys–Cys pairs

The tool reports both reduced and oxidized interpretations so you can compare assumptions about redox state.

How to use the calculator

1) Paste your sequence

Paste a protein sequence using one-letter amino acid codes. The calculator ignores spaces and line breaks.

2) Optionally enter disulfide bonds

If you already know the number of disulfide bonds in your folded protein, enter it directly. If left blank, the calculator assumes the maximum possible value based on cysteine count (floor(Cys/2)).

3) Optionally add molecular weight and concentration

Adding molecular weight allows conversion from molar to mass extinction coefficient. Adding concentration and path length enables predicted A280 values.

Interpreting your output

• ε reduced: assumes no disulfide contribution.
• ε oxidized (selected): includes your entered (or inferred) disulfide count.
• ε oxidized (max): assumes all available cysteines are paired.
• Mass extinction coefficient: useful when concentration is in mg/mL.
• Predicted absorbance: estimated A280 for your entered µM concentration and path length.

When sequence-based estimates can differ from experiments

Sequence-derived ε values are usually excellent for planning and routine quantification, but real spectra can vary due to local environment, partial unfolding, chromophore interactions, buffer background, light scattering, and contaminants such as nucleic acids.

For high-accuracy work (e.g., biophysical characterization, regulatory assays, therapeutic proteins), validate by orthogonal methods: amino acid analysis, dry mass, refractive index methods, or calibrated absorbance standards.

Practical lab tips

• Use a clean baseline buffer matched to your sample formulation.
• Check for turbidity or aggregation; scattering inflates apparent absorbance.
• Confirm path length, especially on microvolume instruments.
• If your construct includes tags/linkers, compute ε for the full expressed sequence.
• Record redox conditions; disulfide status matters for final ε values.

Quick FAQ

Does free cysteine contribute?

In this approximation, only disulfide-bonded cysteines contribute via the +125 term.

Can I use this for peptides?

Yes, if the sequence contains W/Y (and potentially disulfides). For peptides lacking aromatic residues, UV280 is often insensitive.

What units does ε use?

M^-1cm^-1 for molar extinction coefficient; mL·mg^-1·cm^-1 for mass extinction coefficient.