Extinction coefficient calculator

Calculate the molar extinction coefficient at 280 nm for protein concentration determination.

Docs

Input

FASTA input

Cysteine assumption

A280

Path length (cm)

Dilution factor

Concentration basis

Output

Configure input settings on the left, then click "Generate"

What is an extinction coefficient?

The molar extinction coefficient ( $\varepsilon$ ) describes how strongly a protein absorbs light at a given wavelength. At 280 nm, sequence-based protein estimates come mainly from tryptophan, tyrosine, and cystine, the disulfide-bonded form of cysteine.

This calculator uses the protein sequence to estimate $\varepsilon_{280}$ , molecular weight, predicted $A_{280}$ for a 0.1% solution, and optional concentration from a measured absorbance value. It is intended for purified proteins without additional UV-absorbing cofactors, labels, nucleic acids, or other chromophores.

For a broader physicochemical profile, use Protein Parameters, which reports molecular weight, pI, amino acid composition, instability index, GRAVY, and related sequence properties.

How the calculation works

The calculation follows the Pace et al. 1995 280 nm coefficients used by common protein-parameter tools:

\varepsilon_{280} = (n_{Trp} \times 5500) + (n_{Tyr} \times 1490) + (n_{Cystine} \times 125)

Term	Meaning	Contribution
$n_{Trp}$	Number of tryptophan residues (W)	5,500 $\text{M}^{-1}\text{cm}^{-1}$

Free reduced cysteine contributes 0 at 280 nm. A disulfide-bonded cysteine pair contributes 125, so cystine is counted per disulfide bond rather than per cysteine residue.

Molecular weight is calculated from average residue masses, subtracting peptide-bond water and adding terminal water. The Abs 0.1% value is calculated as:

A_{280}^{0.1\%} = \varepsilon_{280} / MW

where $MW$ is molecular weight in daltons. A 0.1% protein solution is 1 mg/mL, or 1 g/L.

Input format

Paste one or more canonical protein sequences as FASTA:

Text

>Protein1
MKWVTFISLLFLFSSAYSRGVFRRDAHKSEVAHRFKDLGE
>Protein2
GIVEQCCTSICSLYQLENYCN

Plain one-letter protein sequence input is also accepted for a single sequence. Uploaded files can use .fasta, .fa, .fas, .txt, or .csv.

CSV input is supported when the header includes a recognizable sequence column such as sequence, seq, or protein:

csv

id,sequence
p1,MKWVTFISLLFLFSSAYSRGVFRRDAHKSEVAHRFKDLGE
p2,GIVEQCCTSICSLYQLENYCN

Only the 20 canonical amino acid letters are accepted:

Text

ARNDCQEGHILKMFPSTWYV

Whitespace and line numbers are ignored. Punctuation, alignment gaps, stop codons, and noncanonical residue codes such as B, J, O, U, X, and Z are rejected instead of being silently removed.

Settings

Setting	Default	What it does
Cysteine assumption	Reduced + oxidized	Returns both reduced and oxidized coefficients by default.
Disulfide bonds	0	Used only when Specified disulfide count is selected. Values above the possible number of cysteine pairs are clamped.
A280	Empty	Optional measured absorbance at 280 nm. When provided, concentration columns are added.
Path length	1 cm	Cuvette or plate path length used in the Beer-Lambert calculation.
Dilution factor	1	Multiplies the calculated concentration to account for sample dilution before measurement.
Concentration basis	Oxidized coefficient	Selects which coefficient is used for the primary concentration columns.

If Specified disulfide coefficient is selected as the concentration basis without using Specified disulfide count as the cysteine assumption, the calculator falls back to the oxidized coefficient.

Results

The standard result table includes one row per sequence:

Column	Description
Protein ID	FASTA header, CSV ID, or generated sequence name.
Amino acids	Sequence length after whitespace and digit cleanup.
Molecular weight (Da)	Average molecular weight for the protein sequence.
Trp count, Tyr count, Cys count	Residue counts used by the 280 nm calculation.
Disulfides assumed	Number of disulfide bonds used for the selected cysteine assumption.
Ext. coeff. reduced	$\varepsilon_{280}$ with all cysteine residues treated as reduced thiols.
Ext. coeff. oxidized	$\varepsilon_{280}$ with all possible cysteine pairs treated as disulfide bonds.
Abs 0.1% reduced / oxidized	Predicted for 1 mg/mL protein using each coefficient.

Optional columns are only shown when they have data:

When	Additional columns
Specified disulfide count is selected	Specified-disulfide extinction coefficient and Abs 0.1%.
A positive A280 value is entered	Concentration in M, uM, and mg/mL for the selected basis.
A positive A280 value is entered	Reduced and oxidized concentration columns for comparison.
A positive A280 value is entered with specified disulfides	Specified-disulfide concentration columns.

The concentration calculation is:

c = (A_{280} / (\varepsilon \cdot l)) \times d

where $c$ is molar concentration, $A_{280}$ is measured absorbance, $\varepsilon$ is the selected extinction coefficient, $l$ is path length in cm, and $d$ is dilution factor.

Warnings and interpretation

Warnings are shown above the result table when a result needs extra context:

Warning	Meaning
No tryptophan residues	The estimate is less reliable because tyrosine and cystine absorb more weakly and are more environment-sensitive.
Odd cysteine count	The oxidized coefficient uses only complete disulfide pairs, so one cysteine remains unpaired.
Custom disulfide count was clamped	The requested count exceeded `floor(Cys / 2)`, the maximum possible number of cysteine pairs.
CSV warnings	Some CSV rows or columns required cleanup or could not be interpreted as sequence records.

The reduced and oxidized values are theoretical assumptions, not structural predictions. The calculator does not infer real disulfide bonding from a structure or sequence motif.

Accuracy considerations

Sequence-based extinction coefficients work best for purified proteins whose 280 nm absorbance is dominated by Trp and Tyr. Results can differ from experimental values because of folding state, pH, buffer composition, detergents, reducing agents, cofactors, prosthetic groups, nucleic acid contamination, labels, post-translational modifications, aggregation, and light scattering.

For quantitative assays, blank the instrument with the matching buffer, measure within the detector's linear range, and use the same cysteine assumption that matches the sample state. For high-accuracy work, determine the extinction coefficient empirically under the assay conditions.

Reference

Pace CN, Vajdos F, Fee L, Grimsley G, Gray T. "How to measure and predict the molar absorption coefficient of a protein." Protein Science 4, 2411-2423 (1995).

Related tools

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GRAVY

Calculate the GRAVY (Grand Average of Hydropathy) score of protein sequences. Positive values indicate hydrophobic proteins, negative values indicate hydrophilic proteins.

Instability Index

Calculate the instability index of protein sequences. Values above 40 indicate an unstable protein with a short half-life in vitro.

Protein molecular weight calculator

Calculate protein molecular weight (MW) from amino acid sequences in Daltons and kilodaltons. Supports FASTA and CSV sequence input, average or monoisotopic masses, initiator Met removal, and disulfide correction.

pI Calculator

Calculate the theoretical isoelectric point (pI) of protein sequences. The pI is the pH at which a protein carries no net electrical charge.

Amino acid composition

Analyze amino acid composition of protein sequences. The tool accepts FASTA sequences and outputs the percentage of each amino acid in the sequence.

Protein motif scanner

Scan protein sequences for biologically important motifs including glycosylation sites, phosphorylation sites, nuclear localization signals, prenylation motifs, and more.

Aggrescan3D

Faithful static-mode Aggrescan3D tool for per-residue aggregation propensity analysis from a single protein structure.

Protein charge plot

Plot net charge vs pH for protein sequences. Visualize how protein charge changes across pH 0-14 and identify the isoelectric point (pI) where the net charge crosses zero.

What is an extinction coefficient?

For a broader physicochemical profile, use Protein Parameters, which reports molecular weight, pI, amino acid composition, instability index, GRAVY, and related sequence properties.

How the calculation works

The calculation follows the Pace et al. 1995 280 nm coefficients used by common protein-parameter tools:

\varepsilon_{280} = (n_{Trp} \times 5500) + (n_{Tyr} \times 1490) + (n_{Cystine} \times 125)

Term	Meaning	Contribution
$n_{Trp}$	Number of tryptophan residues (W)	5,500 $\text{M}^{-1}\text{cm}^{-1}$

Free reduced cysteine contributes 0 at 280 nm. A disulfide-bonded cysteine pair contributes 125, so cystine is counted per disulfide bond rather than per cysteine residue.

Molecular weight is calculated from average residue masses, subtracting peptide-bond water and adding terminal water. The Abs 0.1% value is calculated as:

A_{280}^{0.1\%} = \varepsilon_{280} / MW

where $MW$ is molecular weight in daltons. A 0.1% protein solution is 1 mg/mL, or 1 g/L.

Input format

Paste one or more canonical protein sequences as FASTA:

Text

>Protein1
MKWVTFISLLFLFSSAYSRGVFRRDAHKSEVAHRFKDLGE
>Protein2
GIVEQCCTSICSLYQLENYCN

Plain one-letter protein sequence input is also accepted for a single sequence. Uploaded files can use .fasta, .fa, .fas, .txt, or .csv.

CSV input is supported when the header includes a recognizable sequence column such as sequence, seq, or protein:

csv

id,sequence
p1,MKWVTFISLLFLFSSAYSRGVFRRDAHKSEVAHRFKDLGE
p2,GIVEQCCTSICSLYQLENYCN

Only the 20 canonical amino acid letters are accepted:

Text

ARNDCQEGHILKMFPSTWYV

Settings

Setting	Default	What it does
Cysteine assumption	Reduced + oxidized	Returns both reduced and oxidized coefficients by default.
Disulfide bonds	0	Used only when Specified disulfide count is selected. Values above the possible number of cysteine pairs are clamped.
A280	Empty	Optional measured absorbance at 280 nm. When provided, concentration columns are added.
Path length	1 cm	Cuvette or plate path length used in the Beer-Lambert calculation.
Dilution factor	1	Multiplies the calculated concentration to account for sample dilution before measurement.
Concentration basis	Oxidized coefficient	Selects which coefficient is used for the primary concentration columns.

Results

The standard result table includes one row per sequence:

Column	Description
Protein ID	FASTA header, CSV ID, or generated sequence name.
Amino acids	Sequence length after whitespace and digit cleanup.
Molecular weight (Da)	Average molecular weight for the protein sequence.
Trp count, Tyr count, Cys count	Residue counts used by the 280 nm calculation.
Disulfides assumed	Number of disulfide bonds used for the selected cysteine assumption.
Ext. coeff. reduced	$\varepsilon_{280}$ with all cysteine residues treated as reduced thiols.
Ext. coeff. oxidized	$\varepsilon_{280}$ with all possible cysteine pairs treated as disulfide bonds.
Abs 0.1% reduced / oxidized	Predicted for 1 mg/mL protein using each coefficient.

Optional columns are only shown when they have data:

When	Additional columns
Specified disulfide count is selected	Specified-disulfide extinction coefficient and Abs 0.1%.
A positive A280 value is entered	Concentration in M, uM, and mg/mL for the selected basis.
A positive A280 value is entered	Reduced and oxidized concentration columns for comparison.
A positive A280 value is entered with specified disulfides	Specified-disulfide concentration columns.

The concentration calculation is:

c = (A_{280} / (\varepsilon \cdot l)) \times d

where $c$ is molar concentration, $A_{280}$ is measured absorbance, $\varepsilon$ is the selected extinction coefficient, $l$ is path length in cm, and $d$ is dilution factor.

Warnings and interpretation

Warnings are shown above the result table when a result needs extra context:

Warning	Meaning
No tryptophan residues	The estimate is less reliable because tyrosine and cystine absorb more weakly and are more environment-sensitive.
Odd cysteine count	The oxidized coefficient uses only complete disulfide pairs, so one cysteine remains unpaired.
Custom disulfide count was clamped	The requested count exceeded `floor(Cys / 2)`, the maximum possible number of cysteine pairs.
CSV warnings	Some CSV rows or columns required cleanup or could not be interpreted as sequence records.

The reduced and oxidized values are theoretical assumptions, not structural predictions. The calculator does not infer real disulfide bonding from a structure or sequence motif.

Accuracy considerations

Reference

Pace CN, Vajdos F, Fee L, Grimsley G, Gray T. "How to measure and predict the molar absorption coefficient of a protein." Protein Science 4, 2411-2423 (1995).