molar extinction coefficient calculator protein

Protein ε₂₈₀ Calculator

Estimate the molar extinction coefficient at 280 nm from sequence composition (Trp, Tyr, and cystine), then optionally compute protein concentration from measured absorbance.

Protein sequence (optional, one-letter amino acid code)

Non-amino-acid characters are ignored automatically.

Residue counts used for calculation

Tryptophan count (W)

Tyrosine count (Y)

Cystine count (disulfide pairs)

If you analyzed sequence, this is set to floor(Cys/2). Adjust if your redox state is known.

Optional concentration from A280

Absorbance at 280 nm (A280)

Path length (cm)

Molecular weight (Da, optional)

What is the molar extinction coefficient for proteins?

The molar extinction coefficient (also called molar absorptivity), usually written as ε, describes how strongly a molecule absorbs light at a specific wavelength. For proteins, the most common wavelength is 280 nm, because aromatic residues absorb UV light there.

In practice, ε₂₈₀ lets you convert spectrophotometer absorbance into concentration using the Beer–Lambert law:

A = ε × c × l

A = absorbance (unitless)
ε = molar extinction coefficient (M^-1cm^-1)
c = concentration (M)
l = path length (cm)

Protein ε₂₈₀ equation used in this calculator

This page uses the classic sequence-based estimate:

ε₂₈₀ = (5500 × n_Trp) + (1490 × n_Tyr) + (125 × n_cystine)

where:

n_Trp = number of tryptophan residues (W)
n_Tyr = number of tyrosine residues (Y)
n_cystine = number of disulfide-bonded cysteine pairs

This estimate is widely used for purified proteins and is generally reliable for quick concentration work, especially when a measured reference standard is not available.

How to use this molar extinction coefficient calculator

Method 1: Sequence-based workflow

Paste your one-letter amino acid sequence.
Click Analyze Sequence to auto-count W, Y, and Cys.
Review/adjust cystine count depending on oxidation state.
Click Calculate to get ε₂₈₀.

Method 2: Manual count workflow

Enter residue counts directly (W, Y, cystine pairs).
Click Calculate.

Optional concentration calculation

If you also provide A280 and path length, the calculator returns concentration in:

M (molar)
µM (micromolar)
mg/mL (if molecular weight is entered)

Worked example

Suppose your protein has 2 Trp, 6 Tyr, and 1 disulfide bond:

ε₂₈₀ = (5500 × 2) + (1490 × 6) + (125 × 1) = 20,065 M^-1cm^-1

If A280 = 0.90 in a 1 cm cuvette:

c = A / (εl) = 0.90 / (20,065 × 1) = 4.49 × 10^-5 M = 44.9 µM

Important accuracy notes

Redox state matters: reduced cysteines do not contribute as cystine pairs.
Buffer additives: some reagents absorb in UV and can bias A280.
Nucleic acid contamination: DNA/RNA strongly absorb UV and can inflate apparent protein concentration.
Path length correction: microvolume instruments often use path lengths other than 1 cm.
Protein context: this is a sequence-based estimate, not a direct experimental measurement.

When should you verify experimentally?

For routine purification, sequence-based ε₂₈₀ is often sufficient. For high-precision applications (kinetics, formulation, biopharma release, or quantitative biophysical studies), confirm concentration with orthogonal methods such as amino acid analysis, quantitative colorimetric assays, or calibrated standards.

FAQ

Does every cysteine contribute to A280?

No. The equation uses cystine (disulfide-bonded pairs), not free cysteine residues.

Can I use this for peptides?

Yes, if aromatic residues are present. Very short peptides with no Trp/Tyr can have very low A280 and poor sensitivity.

What if my calculated ε is zero?

That usually means no Trp, Tyr, or cystine contribution was entered. In such cases, A280 may not be an effective quantitation method.

Protein ε280 Calculator