Protein Extinction Coefficient

About Protein Extinction Coefficient

This tool estimates a protein's molar extinction coefficient at 280 nm from its tryptophan, tyrosine and cysteine content, and the absorbance a 1 g/L solution would give. Those numbers let you convert an A280 reading into a concentration without running a colorimetric assay, provided you know the sequence.

How to use it

1. Paste the one-letter protein sequence into the Protein sequence box.
2. Check the residue and Dalton badges to confirm the sequence was read correctly.
3. Read the reduced and cystine-formed extinction coefficients, and the A280 of a 1 g/L solution for each state.
4. Use Copy results to keep the numbers, including the W, Y and C counts.

A 69-residue albumin-like fragment

MKWVTFISLLFLFSSAYSRGVFRRDTHKSEIAHRFKDLGEEHFKGLVLIAFSQYLQQCPFEDHVKLVNE

1 Trp, 2 Tyr, 1 Cys. 8192.5 Da. Extinction coefficient 8480 in both states. A280 of 1 g/L is 1.035

When to use it

• Working out the protein concentration of a purified sample from a NanoDrop A280 reading.
• Predicting whether a protein has enough aromatic residues to be detected reliably at 280 nm.
• Checking how much disulfide formation changes the extinction coefficient.
• Recording the extinction coefficient in the methods for a purification protocol.

Questions about Protein Extinction Coefficient

What formula is used?

The Pace 1995 composition method, the same as ExPASy ProtParam: 5500 times the tryptophans plus 1490 times the tyrosines plus 125 times the cystines. The 1 g/L absorbance is that value divided by the molecular weight.

What is the difference between the two results?

The reduced value treats every cysteine as free. The cystine value assumes cysteines pair up into as many disulfide bonds as possible, one bond per two cysteines, each adding 125.

How do I turn an A280 reading into a concentration?

For a 1 cm path, divide your absorbance by the A280 of a 1 g/L solution to get grams per liter. An absorbance of 0.52 with a factor of 1.035 means roughly 0.50 g/L.

When is this estimate unreliable?

For proteins with no Trp or Tyr, where absorbance is tiny, for samples contaminated with nucleic acid, and for cofactor-bound or unusually folded proteins whose aromatic residues absorb differently.

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