18,575
M^-1 cm^-1
0.3715
AU
0.00002
M
0.3715
AU
18,575
M^-1 cm^-1
0.3715
AU
0.00002
M
0.3715
AU
The Extinction Coefficient Calculator estimates the molar extinction coefficient of a protein at 280 nm using the Pace method. This value (ε280) is essential for UV spectrophotometric protein quantification — the most convenient and non-destructive method for measuring protein concentration. The calculation is based on the number of tryptophan, tyrosine, and cystine (disulfide bond) residues in the protein, which are the primary chromophores absorbing at 280 nm.
The Pace method calculates ε280 from the amino acid composition:
ε280 = nW × 5500 + nY × 1490 + nC × 125
Where:
Once you know ε280, use Beer-Lambert law to calculate concentration: C = A280 / (ε × l), where l is the path length (usually 1 cm).
Inputs
Results
A protein with 2 Trp, 5 Tyr, and 1 disulfide bond has ε280 = 18,575 M⁻¹cm⁻¹. At A280 = 1.0, the concentration would be 1/18575 M = 53.8 µM.
Inputs
Results
A protein rich in aromatic residues (8 Trp, 10 Tyr, 4 disulfides) has a high extinction coefficient of 59,400 M⁻¹cm⁻¹.
Cysteine (free thiol, -SH) does not significantly absorb at 280 nm and is not included. Cystine is formed when two cysteines form a disulfide bond (-S-S-), which contributes a small absorption at 280 nm (ε = 125 M⁻¹cm⁻¹). Count the number of disulfide bonds, not individual cysteine residues. If your protein has 4 cysteines forming 2 disulfide bonds, enter nC = 2.
Proteins without Trp or Tyr have negligible absorbance at 280 nm, making direct UV quantification unreliable. In this case, use alternative methods: A205 measurement (peptide bond absorption), Bradford or BCA colorimetric assays, or measure at 214 nm. Some proteins without aromatic residues can still be quantified using the absorbance at 205 nm, where peptide bonds absorb.
Under denaturing conditions (6 M guanidinium chloride), the Pace method is typically accurate to within ±5%. Under native (folded) conditions, accuracy decreases to about ±10% because the local environment of aromatic residues (burial, hydrogen bonding, nearby charges) can shift their absorption properties. For highest accuracy, experimentally determine ε280 using amino acid analysis or a dry-weight method.
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