Protein Concentration Calculator

How to Use the Protein Concentration Calculator

1. A280 / Beer-Lambert tab: Enter your A280 reading, the molar extinction coefficient (ε), and pathlength. Optionally enter MW for mg/mL output and dilution factor if your sample was diluted.
2. E1% tab: If you have the specific absorbance (E1%) from a datasheet, enter A280, E1%, and pathlength to get mg/mL directly.
3. Standard Curve tab: For BCA or Bradford assays, enter the slope and intercept from your standard curve and the sample absorbance.
4. Click Calculate Concentration and read results in µM and mg/mL.

Beer-Lambert Law for Protein Quantification

The Beer-Lambert law states that the absorbance (A) of a solution is proportional to the concentration (C) and pathlength (l): A = ε × C × l

Rearranging for concentration: C (mol/L) = A / (ε × l)

To convert to mg/mL: multiply the molar concentration by the molecular weight (Da) and divide by 1000.

This method works best for pure protein samples. Nucleic acid contamination (A260 > A280) will cause an overestimate of protein concentration. A260/A280 ratio above 0.6 indicates significant nucleic acid contamination.

A280 vs BCA vs Bradford — Which to Use?

• A280 (direct UV): Fast, non-destructive, no reagents needed. Best for pure proteins with known ε. Not suitable for samples with nucleic acid contamination or highly coloured buffers.
• BCA assay: Colorimetric, very sensitive (~1–2000 µg/mL range). Compatible with reducing agents. Best for complex mixtures and impure samples.
• Bradford assay: Very fast and cheap. Sensitive (~1–500 µg/mL). Incompatible with detergents (Triton, SDS). Best for quick total protein estimates.