Protein Concentration Calculator
Estimate protein concentration from UV absorbance using the Beer – Lambert relationship, a protein-specific molar extinction coefficient, molecular weight, pathlength, and dilution factor.
Measurement inputs
Choose a preset or select Custom protein to enter your own constants.
Molar absorptivity at the measurement wavelength.
Mass of one mole of the selected protein.
Unitless spectrophotometer reading after blank correction.
Optical path through the sample; standard cuvettes are often 1 cm.
Use 1 for undiluted stock, 2 for 1:2, 10 for 1:10, and so on.
Live results
Protein concentration
Mass concentration in the original stock after applying the dilution factor.
Molar concentration
Measured sample concentration
Concentration in µg/mL
Mass absorptivity
Protein reference constants
| Substance | Molecular weight (g/mol) | Extinction coefficient (M⁻¹ cm⁻¹) |
|---|
Preset constants are convenient starting points. For critical work, use the coefficient and molecular weight documented for your exact protein construct, wavelength, buffer, and assay conditions.
How to use this protein concentration calculator
What this calculator does
This calculator estimates the mass concentration of a protein solution from a UV absorbance measurement. It rearranges the Beer – Lambert relationship, A = εbc, to obtain molar concentration and then multiplies by molecular weight and the dilution factor. The result is useful for checking purified-protein stocks, planning loading amounts, normalizing samples before electrophoresis, and documenting a dilution-corrected estimate. It does not replace a validated assay protocol, blank correction, instrument qualification, or judgment about whether your absorbance lies in the linear range.
When to use it
Use it after measuring a reasonably pure protein at its selected wavelength, commonly 280 nm for proteins containing aromatic residues. It is also useful when comparing a stock with a diluted measurement, converting a molar result into mg/mL, or testing how a different pathlength changes the inferred concentration. For mixtures, turbid samples, nucleic-acid contamination, or proteins with uncertain extinction coefficients, consider an orthogonal assay such as Bradford or BCA.
How to calculate
- Select the Protein. A preset fills the extinction coefficient and molecular weight; choose Custom protein when your construct has documented values.
- Enter the Extinction coefficient in M⁻¹ cm⁻¹ and the Molecular weight in g/mol. Both must be positive finite numbers.
- Enter the blank-corrected Absorbance at λmax. The accepted format uses a period as the decimal separator; scientific notation and ambiguous decimal-comma entries are rejected.
- Enter the optical Pathlength in centimeters and the Dilution factor. Use 1 for an undiluted sample and 10 for a 1:10 measurement.
- Read the live results. Use Reset to restore the IgG example or Download Excel to save the current inputs and calculated values in a validated workbook.
Input guide
Protein is a required preset selector. It supplies protein-specific constants; changing it can alter concentration even when absorbance is unchanged. Extinction coefficient is required, measured in M⁻¹ cm⁻¹, and represents how strongly the analyte absorbs light. A realistic IgG value is 210,000. Do not enter a mass extinction coefficient into this molar field. Molecular weight is required in g/mol; 150,000 is a common approximate value for IgG. Use the value for the actual sequence and oligomeric state.
Absorbance at λmax is a required unitless optical-density reading. The default worked example uses 280 to mirror a simple formula demonstration, although practical spectrophotometer readings are commonly much smaller; always enter the observed absorbance, not the wavelength. Pathlength is required in centimeters; 1 cm is common for a standard cuvette, while microvolume instruments may use a shorter effective path. A shorter path with the same absorbance implies a higher concentration. Dilution factor is required and must be at least 1; it scales the measured-sample concentration back to the original stock. A frequent mistake is entering 0.1 for a 1:10 dilution instead of 10.
Output guide
Protein concentration is the dilution-corrected mass concentration in mg/mL. Molar concentration is the stock concentration in mol/L before mass conversion. Measured sample concentration is the mass concentration in the diluted cuvette or measurement vessel, before multiplying by dilution factor. Concentration in µg/mL is the same stock mass concentration expressed in micrograms per milliliter. Mass absorptivity equals the molar extinction coefficient divided by molecular weight and is shown in mL·mg⁻¹·cm⁻¹. Zero absorbance produces zero concentration; unusually high results should prompt a check of dilution, units, blank correction, and instrument linearity.
Worked example
For IgG, use ε = 210,000 M⁻¹ cm⁻¹, molecular weight = 150,000 g/mol, absorbance = 280, pathlength = 1 cm, and dilution factor = 10. Molar concentration in the measured sample is 280 ÷ (210,000 × 1) = 0.001333333 mol/L. Multiplying by 150,000 g/mol gives 200 g/L, numerically equal to 200 mg/mL. Applying the tenfold dilution factor gives 2,000.000 mg/mL, or 2,000,000 µg/mL. The workbook export preserves the canonical numeric values rather than copying rounded screen text.
Learn more
The Beer – Lambert law overview from Chemistry LibreTexts explains the relationship among absorbance, pathlength, concentration, and molar absorptivity. Harvard Medical School's protein concentration measurement guide discusses UV-based protein quantification and the need for an extinction coefficient. For sequence-derived properties, the Swiss Institute of Bioinformatics provides ExPASy ProtParam. For a complementary dye-binding method, see the Bio-Rad Bradford assay guide.