Beer-Lambert, rearranged for concentration
Absorbance is proportional to concentration (A = ε · b · c), so molar concentration is the 280 nm reading divided by the molar extinction coefficient ε and the path length b (usually 1 cm). Multiply by the dilution factor if you diluted before reading. To get mg/mL, multiply the molar result by the protein's molecular weight and divide by 1000.
Extinction coefficients for common proteins
| Protein | MW (kDa) | A280 of 1 mg/mL |
|---|---|---|
| BSA (albumin) | 66.5 | 0.66 |
| IgG antibody | 150 | 1.35 |
| Lysozyme | 14.3 | 2.64 |
| Ribonuclease A | 13.7 | 0.71 |
The last column is the mass extinction coefficient: divide your blank-corrected A280 by it to get mg/mL directly. From a sequence you can compute ε as (5500 × Trp) + (1490 × Tyr) + (125 × disulfides).
Getting a clean reading
- Blank first. Zero the instrument on buffer alone, then subtract that blank so only the protein contributes to A280.
- Keep A between 0.1 and 1.0. Outside that band the reading is either noise or a saturated detector. Dilute and rescale by the dilution factor when you go over.
- Watch for A260 contamination. Nucleic acids absorb near 260 nm and spill into 280, inflating your result. A 260/280 ratio well below 0.6 suggests a clean protein sample.
Common questions
How does A280 measure protein concentration?
Proteins absorb 280 nm light through their tryptophan and tyrosine residues. Beer-Lambert says absorbance equals the extinction coefficient times path length times concentration, so concentration is the absorbance divided by that product.
Why should the absorbance reading stay between 0.1 and 1.0?
Below 0.1 the signal is small and noisy. Above about 1.0 the detector saturates and the linear Beer-Lambert relationship breaks down. If your reading is over 1.0, dilute the sample and multiply the result back by the dilution factor.
When should I not use A280?
A280 needs a pure protein of known extinction coefficient. It is unreliable for proteins low in tryptophan and tyrosine, and it reads high when nucleic acids, pigments or other 280 nm absorbers are present. Use Bradford or BCA with a BSA standard in those cases.
