Calculator
Formula Used
Molar extinction coefficient: ε280 = (W × 5500) + (Y × 1490) + (cystine pairs × 125)
Corrected absorbance: A = (sample A280 - blank A280) × dilution factor
Molar concentration: c = A / (ε280 × path length)
Mass concentration: mg/mL = molar concentration × molecular weight in Da
Mass extinction coefficient: εmass = ε280 / molecular weight in Da
How to Use This Calculator
Choose sequence mode when you have a full protein sequence. Paste the one-letter sequence into the textarea. Choose manual count mode when you already know W, Y, and C counts. Enter molecular weight, absorbance, blank value, dilution, path length, and purity. Select the cystine option that matches your sample state. Press Calculate to show results above the form. Use CSV or PDF buttons to save the report.
Example Data Table
| Sample | W | Y | Cystine pairs | MW Da | A280 | ε280 | mg/mL |
|---|---|---|---|---|---|---|---|
| Protein A | 5 | 12 | 3 | 52000 | 0.78 | 45755 | 0.886 |
| Protein B | 2 | 8 | 0 | 33000 | 0.52 | 22920 | 0.749 |
| Protein C | 9 | 24 | 5 | 120000 | 1.20 | 85885 | 1.677 |
Understanding Protein Extinction Coefficients
A protein extinction coefficient links light absorption to protein amount. It is usually reported at 280 nm, because tryptophan, tyrosine, and cystine absorb strongly there. This value helps a lab estimate concentration from a UV spectrophotometer reading. It also helps compare purified batches, check dilution plans, and prepare assay stocks without repeated colorimetric tests.
Why Residues Matter
The calculation starts with residue counts. Tryptophan has the largest effect. Tyrosine adds a smaller effect. Oxidized cysteine pairs, called cystines, add a small contribution. Reduced cysteine is normally ignored at 280 nm. A sequence based method is useful when the full amino acid sequence is known. A manual count method is useful for fragments, engineered constructs, or proteins with edited residue summaries.
Using Absorbance Data
Beer Lambert law connects absorbance, path length, concentration, and extinction coefficient. A standard cuvette often uses a 1 cm path. Microvolume instruments may use shorter effective paths. This calculator lets you enter the path length, blank absorbance, dilution factor, molecular weight, and purity. It then reports molar concentration, mass concentration, and purity adjusted target concentration. These outputs support buffer exchange, enzyme setup, antibody dilution, and general chemistry reporting.
Practical Notes
Good results need clean inputs. Use the mature protein sequence when signal peptides are removed. Include tags when they remain in the measured sample. Use oxidized cystine settings only when disulfide bonds are expected. If the protein is denatured or reduced, choose the reduced setting. Very low absorbance values can create unstable concentration estimates. Read the blank with the same buffer, cell, and wavelength.
Interpreting Results
The molar coefficient is best for molar concentration work. The mass coefficient is convenient for mg per mL reporting. The absorbance for 1 mg per mL is useful during quick checks. Exported CSV and PDF reports keep the assumptions beside the result. That makes the calculation easier to review later, especially when several constructs or purification fractions are compared in one lab notebook.
Quality Limits
The value is still an estimate. Post translational changes, bound cofactors, nucleic acid carryover, turbidity, and scattering can change absorbance. For critical release testing, confirm concentration with an independent validated method and documented instrument calibration records before making final batch release decisions.
FAQs
What is a protein extinction coefficient?
It is a value that links absorbance to concentration. For proteins, it is commonly calculated at 280 nm from tryptophan, tyrosine, and cystine content.
Why does tryptophan matter most?
Tryptophan absorbs strongly at 280 nm. A small number of tryptophan residues can greatly raise the molar extinction coefficient.
Should I use cysteine or cystine?
Use cystine when cysteine residues form disulfide bonds. Use reduced mode when the sample is reduced and cysteines are not paired.
Can I paste a FASTA sequence?
Yes. The calculator removes non-letter characters. For best results, remove headers, spaces, and unrelated sequence notes before calculating.
What path length should I enter?
Use the instrument path length. Standard cuvettes often use 1 cm. Microvolume devices may use a shorter effective path.
What does mass extinction coefficient mean?
It converts absorbance into mg per mL. It is calculated by dividing the molar coefficient by molecular weight in daltons.
Why enter a blank absorbance?
The blank corrects buffer, cell, and baseline absorption. It should be measured with the same buffer and wavelength.
Are exported reports editable?
The CSV file can be edited in spreadsheet tools. The PDF is meant for quick saving, printing, and lab record attachment.