Beer-Lambert Protein Calculator Formula
Understand the math behind the beer-lambert protein calculator. Each variable explained with a worked example.
Formulas Used
Molar Concentration
conc_molar = a280 / (extinction * path_length)Mass Concentration
conc_mg = a280 / (extinction * path_length) * mw_protein / 1000Variables
| Variable | Description | Default |
|---|---|---|
a280 | Absorbance at 280 nm | 1 |
extinction | Extinction Coefficient (E)(M⁻¹cm⁻¹) | 43000 |
path_length | Path Length(cm) | 1 |
mw_protein | Protein Molecular Weight(Da) | 45000 |
How It Works
Protein Concentration from A280
UV absorbance at 280 nm is widely used to measure protein concentration. Aromatic residues (Trp, Tyr) and disulfide bonds absorb at this wavelength.
Formula
c (M) = A280 / (epsilon × l)
c (mg/mL) = c (M) × MW / 1000
The extinction coefficient epsilon can be calculated from the amino acid sequence using tools like ProtParam. Typical values range from 10,000 to 100,000 M⁻¹cm⁻¹.
Worked Example
A protein with epsilon = 43,000 M⁻¹cm⁻¹ and MW = 45 kDa, measured A280 = 1.0 in a 1 cm cuvette.
- 01c = 1.0 / (43000 × 1) = 2.326 × 10⁻⁵ M = 23.26 uM
- 02c = 23.26 × 10⁻⁶ × 45000 / 1000 = 1.047 mg/mL
Frequently Asked Questions
What if my protein has no tryptophan?
Proteins with no Trp have very low A280 extinction coefficients (only from Tyr and Cys). The measurement becomes less sensitive. Consider using BCA or Bradford assays instead, or measure at 205 nm (peptide bond absorption).
How accurate is A280 measurement?
Very accurate for pure proteins with known extinction coefficients. Contaminating nucleic acids absorb at 260 nm and can be corrected using the A260/A280 ratio. Pure protein has A260/A280 around 0.55-0.6.
What is the A280 0.1% convention?
Some references report E0.1% = absorbance of a 1 mg/mL solution in a 1 cm path. To convert: epsilon (M⁻¹cm⁻¹) = E0.1% × MW / 10.
Ready to run the numbers?
Open Beer-Lambert Protein Calculator