How to calculate protein concentration
Determining the concentration of a purified protein in solution is most commonly done by measuring its absorbance at 280 nm (A₂₈₀). Aromatic amino acids, specifically tryptophan and tyrosine, strongly absorb UV light at this wavelength.
Beer-Lambert Law for proteins
The bedrock behind this calculation is the Beer-Lambert Law, relating the attenuation of light to the properties of the material through which the light is traveling.
A = ε × l × c
Rearranging this to solve for molar concentration (c) yields:
c = A / (ε × l)
- A: Absorbance (Optical Density) at 280 nm
- ε: Molar extinction coefficient (M⁻¹cm⁻¹)
- l: Path length of the cuvette (typically 1 cm)
- c: Molar concentration (M or mol/L)
To convert this molar concentration into a mass concentration (like mg/mL), you multiply the molar concentration by the molecular weight of the protein.
How to use the calculator
Simply enter all parameters known about your protein sample:
- Enter the measured Absorbance at A₂₈₀.
- Input your protein's Extinction Coefficient. If you don't know it, tools like ProtParam can calculate it from your amino acid sequence.
- Enter the Molecular Weight in kDa.
- If you diluted the sample prior to measuring, update the Dilution Factor.