Protein Concentration Calculator
Calculate protein concentration from UV absorbance measurements
Measurement Parameters
Measured Concentration
11.39 µM
0.285 mg/mL
All Units
Beer-Lambert Law
c = A / (ε × l) = 0.5 / (43890 × 1)
About Protein Concentration Measurement
UV absorbance at 280nm is commonly used to determine protein concentration. The method relies on:
- Aromatic residues: Trp, Tyr, and Phe absorb UV light
- Extinction coefficient: Sequence-specific absorption property
- Beer-Lambert law: A = εcl relates absorbance to concentration
For accurate results, ensure absorbance is between 0.1 and 1.0 (linear range).
Protein Concentration Calculator Overview
The protein concentration calculator converts a UV absorbance reading into protein concentration using the Beer-Lambert law. Spectrophotometry at 280 nm (A280) is one of the fastest, most non-destructive ways to quantify a purified protein because the aromatic side chains of tryptophan, tyrosine, and to a small extent phenylalanine and disulfide bonds absorb ultraviolet light. By feeding the measured absorbance, the molar extinction coefficient, the cuvette path length, and the molecular weight into this tool, you instantly get concentration expressed in molar (M), micromolar (µM), nanomolar (nM), and mg/mL.
Unlike colorimetric assays such as Bradford or BCA, the A280 method requires no reagents, no incubation, and no standard curve. You simply blank the spectrophotometer against buffer, read the sample, and let the A280 calculator handle the conversion. Because the calculation is direct, the accuracy of your result depends almost entirely on the accuracy of the extinction coefficient you supply and on keeping the absorbance within the instrument's linear range, ideally between 0.1 and 1.0 absorbance units.
This page also applies an optional dilution factor so that if you diluted a concentrated stock before reading it, the calculator reports both the measured (cuvette) concentration and the back-calculated original stock concentration. That makes it practical for protein purification, recombinant expression, antibody preparation, and enzymology workflows where stock concentrations must be known precisely.
Beer-Lambert Law and the Formula Used
The calculator solves the Beer-Lambert law for concentration. The law states that absorbance is proportional to the concentration of the absorbing species, the path length of light through the sample, and the molar extinction (absorptivity) coefficient:
A = ε × c × l
Rearranging to isolate concentration gives the core equation the page evaluates:
c = A / (ε × l)
Here concentration c is returned in molar units (mol/L). The tool then multiplies by 1,000,000 to obtain micromolar and by 1,000,000,000 to obtain nanomolar. To convert the molar value into a mass concentration in mg/mL, it multiplies the molar concentration by the protein's molecular weight in daltons. Finally, if a dilution factor greater than one is entered, the calculator multiplies the measured concentration by that factor to recover the concentration of the undiluted stock. Every reported number flows directly from these few arithmetic steps, so understanding them lets you sanity-check any result by hand.
Concentration from Absorbance
Where:
- A= Measured absorbance at 280 nm (unitless)
- ε= Molar extinction coefficient (M⁻¹cm⁻¹)
- l= Optical path length of the cuvette (cm)
- c= Molar concentration of protein (mol/L)
- MW= Molecular weight of the protein (Da, g/mol)
- DF= Dilution factor applied to recover the stock concentration
Choosing the Right Extinction Coefficient
The single most important input to this protein quantification tool is the molar extinction coefficient (ε). It is sequence-specific and is dominated by the number of tryptophan and tyrosine residues, with a small contribution from cystines (disulfide-bonded cysteine pairs). A widely used estimate from Gill and von Hippel, implemented in tools such as ExPASy ProtParam, is:
ε (M⁻¹cm⁻¹) = (nTrp × 5500) + (nTyr × 1490) + (ncystine × 125)
| Residue / feature | Contribution to ε (M⁻¹cm⁻¹) |
|---|---|
| Tryptophan (Trp, W) | 5500 per residue |
| Tyrosine (Tyr, Y) | 1490 per residue |
| Cystine (disulfide pair) | 125 per bond |
If a protein contains no tryptophan or tyrosine, it will barely absorb at 280 nm and the A280 method becomes unreliable; in that case a colorimetric assay is the better choice. Always use the extinction coefficient that matches whether your protein is reduced (free cysteines) or oxidized (disulfides formed), because the cystine term shifts ε slightly. Entering an extinction coefficient that is too high makes the calculated concentration too low, and vice versa, so this value deserves careful attention.
Units, mg/mL, and Molar Conversions
The protein concentration calculator reports results in several units so you can use whichever your protocol requires. Molar concentration comes straight from the Beer-Lambert solution. Micromolar (µM) and nanomolar (nM) are scaled versions of that molar value, useful when you need to set up binding reactions, enzyme kinetics, or stoichiometric mixing where mole ratios matter.
Mass concentration in mg/mL is obtained by multiplying the molar concentration by the molecular weight. Because molecular weight is expressed in daltons (g/mol), and molar concentration in mol/L, the product yields g/L, which is numerically identical to mg/mL. This is the unit most often quoted for stock solutions, SDS-PAGE loading, and storage records.
| Output unit | How it is derived |
|---|---|
| Molar (M) | A / (ε × l) |
| Micromolar (µM) | Molar × 1,000,000 |
| Nanomolar (nM) | Molar × 1,000,000,000 |
| mg/mL | Molar × molecular weight (Da) |
Keeping these relationships in mind makes it easy to convert a published mg/mL stock into a working molar concentration for your next experiment without re-measuring anything.
Applying the Dilution Factor
Concentrated protein stocks frequently exceed the linear absorbance range of a spectrophotometer, so it is standard practice to dilute the sample before reading A280. The dilution factor input lets the calculator scale the cuvette reading back up to the true stock concentration. A dilution factor of 10, for example, means one part stock was mixed with nine parts buffer; the tool multiplies the measured concentration by 10 to report the original.
When the dilution factor is left at 1, the measured and original concentrations are identical and the calculator simply reports the cuvette value. When it is greater than 1, the page shows two result panels: the measured concentration in the diluted cuvette and the back-calculated original sample concentration in both µM and mg/mL. This dual readout keeps your records honest and prevents the common mistake of forgetting to account for dilution when documenting a stock.
Choose a dilution that lands the diluted absorbance comfortably between 0.1 and 1.0. Diluting too little leaves you outside the linear range where Beer-Lambert breaks down; diluting too much amplifies pipetting error and instrument noise in the final back-calculated number.
Accuracy, Limitations, and Best Practices
The A280 protein concentration method is fast but has well-known pitfalls. Light scattering from aggregates or particulates inflates the apparent absorbance, so always inspect or centrifuge cloudy samples. Nucleic acid contamination is another classic interferent because DNA and RNA absorb strongly at 260 nm and partly at 280 nm; checking the A260/A280 ratio (ideally near 0.57 for pure protein) flags this problem.
Buffer components that absorb in the UV, such as certain detergents, imidazole, or DTT at high concentrations, can also bias the reading, which is why blanking against the exact sample buffer is essential. Temperature, bubbles in the cuvette, fingerprints on the optical faces, and a mismatched path length all introduce error. For the most reliable quantification, keep absorbance in the 0.1 to 1.0 window, read in triplicate, and confirm the extinction coefficient corresponds to your protein's true amino acid sequence.
When a protein lacks aromatic residues or your sample contains interfering species you cannot remove, switch to a Bradford, BCA, or Lowry assay. Used within its limits, however, this protein quantification calculator delivers reproducible, reagent-free concentration values in seconds.
Worked Examples
Standard A280 reading of a 25 kDa protein
Problem:
A purified protein gives A280 = 0.5 in a 1 cm cuvette. Its extinction coefficient is 43,890 M⁻¹cm⁻¹ and molecular weight is 25,000 Da. Find the concentration.
Solution Steps:
- 1Apply Beer-Lambert: c = A / (ε × l) = 0.5 / (43,890 × 1).
- 2Compute molar concentration: c = 1.139 × 10⁻⁵ M.
- 3Convert to micromolar: 1.139 × 10⁻⁵ × 1,000,000 = 11.39 µM.
- 4Convert to mg/mL: 1.139 × 10⁻⁵ × 25,000 = 0.285 mg/mL.
Result:
Concentration ≈ 11.39 µM, equivalent to about 0.285 mg/mL.
Diluted concentrated stock
Problem:
A concentrated stock was diluted 10-fold before reading. The diluted sample shows A280 = 0.8, ε = 50,000 M⁻¹cm⁻¹, path length 1 cm, MW = 40,000 Da. What is the original stock concentration?
Solution Steps:
- 1Measured molar concentration: c = 0.8 / (50,000 × 1) = 1.6 × 10⁻⁵ M = 16.0 µM.
- 2Measured mass concentration: 1.6 × 10⁻⁵ × 40,000 = 0.64 mg/mL.
- 3Apply the dilution factor of 10 to the measured molar value: 16.0 × 10 = 160 µM.
- 4Apply the dilution factor to mg/mL: 0.64 × 10 = 6.4 mg/mL.
Result:
Original stock ≈ 160 µM, or about 6.4 mg/mL.
Antibody (IgG) quantification
Problem:
An IgG sample reads A280 = 0.42 in a 1 cm cuvette. Using ε = 210,000 M⁻¹cm⁻¹ and MW = 150,000 Da, calculate the concentration in µM and mg/mL.
Solution Steps:
- 1Beer-Lambert: c = 0.42 / (210,000 × 1) = 2.0 × 10⁻⁶ M.
- 2Convert to micromolar: 2.0 × 10⁻⁶ × 1,000,000 = 2.0 µM.
- 3Convert to mg/mL: 2.0 × 10⁻⁶ × 150,000 = 0.30 mg/mL.
- 4With no dilution (factor 1), measured and original values are the same.
Result:
Concentration ≈ 2.0 µM, equivalent to 0.30 mg/mL.
Tips & Best Practices
- ✓Always blank the spectrophotometer against the exact buffer your protein is in.
- ✓Keep absorbance between 0.1 and 1.0 for valid Beer-Lambert results.
- ✓Centrifuge or filter cloudy samples to remove scattering aggregates.
- ✓Check the A260/A280 ratio to catch nucleic acid contamination.
- ✓Use an extinction coefficient computed from the actual amino acid sequence.
- ✓Read each sample in triplicate and average to reduce pipetting error.
- ✓Match the path length entered here to your cuvette (commonly 1 cm).
- ✓Switch to BCA or Bradford if the protein lacks tryptophan and tyrosine.
Frequently Asked Questions
Sources & References
Last updated: 2026-06-05
Help us improve!
How would you rate the Protein Concentration Calculator?
Editorial Note
MyCalcBuddy Editorial Team
This page is maintained as an educational calculator reference.
Formula Source: Standard Mathematical References
by Various