Protein Concentration Calculator
Calculate the concentration of your protein sample from UV absorbance readings. Uses the Beer-Lambert law (A = εlc) for accurate results.
Measured absorbance at 280 nm wavelength
Molar extinction coefficient in M⁻¹cm⁻¹ (use 1 for simplified mode)
Light path length through the sample (typically 1 cm)
If sample was diluted, enter the dilution factor (e.g., 10 for 1:10 dilution)
What is the Beer-Lambert law and how does it apply to protein concentration?
The Beer-Lambert law (A = εlc) relates absorbance to concentration: A is absorbance, ε is molar extinction coefficient (M⁻¹cm⁻¹), l is path length (cm), and c is concentration (M). This law assumes monochromatic light, homogeneous sample, and no fluorescence or scattering. For proteins, absorbance at 280 nm (A280) is measured because aromatic amino acids (tryptophan, tyrosine, phenylalanine) absorb UV light at this wavelength.
How do I find the extinction coefficient for my protein?
You can determine extinction coefficient in three ways: (1) Sequence-based prediction using ExPAS ProtParam - input your amino acid sequence to calculate theoretical ε280, (2) Experimental determination using BCA or Bradford assay with known concentration, (3) Use an estimated average of 1 mg/mL = 1 A280 for rough estimates. For IgG antibodies, ε280 ≈ 210,000 M⁻¹cm⁻¹ is commonly used. Always verify with a standard curve for critical experiments.
What path length should I use?
Standard cuvettes have 1 cm path length, which is the default. Microvolume cuvettes (e.g., NanoDrop) use shorter paths (0.5 mm or less) but have built-in path length correction. If using a plate reader, path length varies by well diameter - use manufacturer software or calculate: actual path = well diameter × sample volume. Always verify your cuvette dimensions before calculating.
Why does my absorbance exceed 1.0? Should I dilute my sample?
Absorbance > 1.0 is in the non-linear range where the Beer-Lambert law breaks down due to excessive light scattering and inner filter effects. For accurate measurements, dilute your sample until A280 is between 0.1-1.0. Then multiply your calculated concentration by the dilution factor. If absorbance is very high (>3), use a shorter path cuvette or different analytical method.