Extinction Coefficient Calculator
Calculate the molar extinction coefficient for proteins (at 280 nm) and oligonucleotides (at 260 nm). Enter a sequence to determine the extinction coefficient, estimate molecular weight, and convert absorbance readings to concentration using the Beer-Lambert law.
Enter a one-letter amino acid sequence. FASTA headers and whitespace are removed automatically.
Each disulfide bond = 2 Cys residues paired. Leave 0 if reducing conditions or unknown.
Enter a single-stranded DNA or RNA sequence (A, T/U, G, C).
What is Extinction Coefficient?
The molar extinction coefficient (also called molar absorptivity), symbolized as epsilon, quantifies how strongly a substance absorbs light at a particular wavelength. It is central to the Beer-Lambert law:
A = epsilon x l x c
Where A is absorbance (unitless), epsilon is the molar extinction coefficient (M-1 cm-1), l is the path length in centimeters (typically 1 cm in a standard cuvette), and c is the molar concentration (M).
Rearranging this equation allows you to determine the concentration of a solution from its absorbance reading: c = A / (epsilon x l). This makes extinction coefficients indispensable for protein and nucleic acid quantification in the laboratory.
How to Calculate for Proteins
Protein extinction coefficients at 280 nm are calculated from the amino acid composition using the method described by Pace et al. (1995). The UV absorbance of proteins at 280 nm is dominated by three amino acids:
| Amino Acid | Contribution at 280 nm | Absorbing Group |
|---|---|---|
| Tryptophan (W) | 5,500 M-1 cm-1 | Indole ring |
| Tyrosine (Y) | 1,490 M-1 cm-1 | Phenol ring |
| Cystine (disulfide bond) | 125 M-1 cm-1 | S-S bond |
epsilon(280) = nW x 5500 + nY x 1490 + nSS x 125
Where nW is the number of tryptophan residues, nY is the number of tyrosine residues, and nSS is the number of disulfide bonds. If a protein contains no Trp, Tyr, or disulfide bonds, it will have negligible absorbance at 280 nm, and alternative quantification methods (e.g., Bradford assay or BCA assay) should be used.
How to Calculate for DNA/RNA
For single-stranded oligonucleotides, the extinction coefficient at 260 nm can be approximated by summing the individual nucleotide contributions. While the nearest-neighbor method accounts for stacking interactions and is more accurate, the individual-base method provides a practical estimate:
| Nucleotide | epsilon at 260 nm (M-1 cm-1) | MW (Da) |
|---|---|---|
| dAMP (A) | 15,400 | 331.2 |
| dTMP (T) | 8,700 | 322.2 |
| dGMP (G) | 11,500 | 347.2 |
| dCMP (C) | 7,400 | 307.2 |
| UMP (U, RNA) | 9,900 | 324.2 |
The total extinction coefficient is the sum of individual contributions. For DNA, a hypochromicity correction factor of approximately 0.9 can be applied to account for base stacking in the oligonucleotide context, though this calculator uses the uncorrected sum for simplicity.
Molecular weight for a single-stranded DNA oligonucleotide is calculated as the sum of individual nucleotide molecular weights minus (N-1) x 61.96 (for water loss during phosphodiester bond formation), plus 17.01 for the 3'-OH and 79.0 for the 5'-phosphate, or simplified as: MW = sum of nucleotide MWs - 61.96 x (N-1).
Applications
Protein Concentration Determination
UV spectrophotometry at 280 nm is the most common method for measuring protein concentration. With a known extinction coefficient, a single absorbance reading provides an accurate concentration value without destructive assays.
DNA/RNA Quantification
Absorbance at 260 nm is standard for nucleic acid quantification. The extinction coefficient converts raw absorbance to molar concentration, enabling precise calculation of moles for cloning, ligation, and transfection experiments.
Purity Assessment (A260/A280)
The ratio of absorbance at 260 nm to 280 nm indicates nucleic acid purity. Pure DNA has an A260/A280 ratio of approximately 1.8, while pure RNA is around 2.0. Protein contamination lowers this ratio.
Stoichiometry and Binding Studies
Accurate extinction coefficients are essential for determining binding stoichiometry in protein-ligand, protein-DNA, and enzyme kinetics experiments where precise molar ratios are required.
Frequently Asked Questions
What if my protein has no tryptophan or tyrosine residues?
Proteins lacking Trp, Tyr, and disulfide bonds will have an extinction coefficient at 280 nm of zero or near-zero. In this case, use alternative methods like the Bradford assay (Coomassie binding), BCA assay, or measure at 205 nm where peptide bonds absorb.
How accurate is the Pace method for extinction coefficients?
The Pace method (Trp x 5500 + Tyr x 1490 + Cystine x 125) is accurate to within approximately 5% for most proteins under denaturing conditions. Under native conditions, the actual value may differ due to the local environment of chromophoric residues and tertiary structure effects.
Should I use the extinction coefficient under native or denaturing conditions?
The Pace method gives the extinction coefficient for the denatured (unfolded) protein. For native conditions, the value may differ by up to 10%. For highest accuracy, measure absorbance in 6 M guanidinium chloride to ensure complete denaturation, then calculate concentration using the denatured epsilon.
Why is A260/A280 ratio important for DNA purity?
The A260/A280 ratio reflects the relative absorbance contributions of nucleic acids (peak at 260 nm) versus proteins (peak at 280 nm). A ratio of 1.8 for DNA or 2.0 for RNA indicates a pure sample. Lower values suggest protein contamination, while higher values may indicate RNA contamination in DNA samples.
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