Protein Extinction Coefficient Calculator (A280)
Estimate the molar extinction coefficient at 280 nm directly from amino acid sequence using the standard Pace method.
What is a protein extinction coefficient?
The protein extinction coefficient tells you how strongly a protein absorbs ultraviolet light, typically at 280 nm. In practical terms, it lets you convert an absorbance reading from a spectrophotometer into concentration. For purified proteins, this is one of the fastest and most common quantification methods in molecular biology, biochemistry, and protein engineering labs.
Most of the absorbance at 280 nm comes from aromatic residues, especially tryptophan (W) and tyrosine (Y). Disulfide-linked cystines also contribute slightly. Because these features can be counted from sequence, a useful approximation of the molar extinction coefficient can be calculated before you even purify the protein.
Formula used in this calculator
This tool uses the classical empirical relationship:
ε280 = 5500 × n(W) + 1490 × n(Y) + 125 × n(cystine)
- n(W) = number of tryptophan residues
- n(Y) = number of tyrosine residues
- n(cystine) = number of disulfide bonds (not free cysteines)
The resulting units are M-1cm-1. If concentration and path length are known, absorbance is estimated with Beer-Lambert law: A = εcl.
Reduced vs oxidized protein
If your sample is strongly reducing (for example, contains DTT or TCEP), most cysteines are not in disulfides. In that case set disulfide bonds to zero. If the protein is secreted or known to contain stable disulfides, enter the expected number of cystine pairs for better accuracy.
How to use the calculator
- Paste a protein sequence in one-letter amino acid code.
- Enter how many disulfide bonds are expected in your folded/oxidized state.
- Optionally enter concentration (µM) and path length to predict A280.
- Optionally provide molecular weight to get mass extinction coefficient.
- Click Calculate to view residue counts, ε280, and derived values.
Example
Suppose your sequence has 2 Trp, 5 Tyr, and 1 disulfide bond. Then:
ε280 = (5500 × 2) + (1490 × 5) + (125 × 1) = 18,575 M-1cm-1
At 20 µM in a 1 cm cuvette, predicted absorbance is: A280 = 18,575 × 20×10-6 × 1 = 0.372
Quick interpretation guide
| Range of ε280 (M-1cm-1) | Typical interpretation |
|---|---|
| < 5,000 | Low aromatic content; UV quantification may be less sensitive. |
| 5,000 – 30,000 | Common range for many soluble enzymes and domains. |
| > 30,000 | High aromatic content or larger proteins; usually strong UV signal. |
Practical lab tips for better A280 quantification
- Blank with the exact buffer used for your protein sample.
- Avoid nucleic acid contamination; DNA/RNA absorb strongly near 260 nm and can skew readings.
- Keep absorbance in the linear instrument range (often around 0.1 to 1.0).
- Use accurate path length, especially in microvolume spectrophotometers.
- Remember that sequence-based values are estimates; validate experimentally when precision matters.
Frequently asked questions
Why is my predicted extinction coefficient near zero?
Proteins with few or no Trp/Tyr residues can have very low absorbance at 280 nm. In such cases, consider alternative quantification methods (BCA, Bradford, amino acid analysis, or absorbance at other wavelengths if relevant).
Should I count all cysteines as disulfides?
No. Only cysteine residues that form disulfide bonds contribute as cystine. Free thiols do not receive the +125 term.
Is this calculator valid for peptides?
Yes, as long as sequence is known. For very short peptides, measurement uncertainty can dominate, so verify with standards.