Calculate protein concentration from UV absorbance at 280nm using the Beer-Lambert law. Includes A260/A280 purity assessment for nucleic acid contamination.
Last updated: March 2026
Protein concentration measurement is fundamental in biochemistry and molecular biology. Accurate quantification is essential for enzyme activity assays, SDS-PAGE loading, Western blotting, protein purification monitoring and standardizing experimental conditions.
The Beer-Lambert law describes the relationship between absorbance, concentration and optical path length: A = ε × c × l. At 280 nm, proteins absorb ultraviolet light primarily because of tryptophan and tyrosine residues, with a smaller contribution from cystine.
This calculator uses a mass extinction coefficient in (mg/mL)⁻¹ cm⁻¹, allowing protein concentration to be calculated directly in mg/mL. Do not enter a molar extinction coefficient expressed in M⁻¹ cm⁻¹.
Common protein quantification methods include direct A280 measurement, the Bradford assay and the BCA assay. A280 is fast and non-destructive, while Bradford and BCA assays use calibration curves prepared from known protein standards.
This calculator requires a mass extinction coefficient. A molar extinction coefficient in M⁻¹ cm⁻¹ cannot be entered directly without conversion.
A280 measurement is most appropriate for purified or partially purified protein samples when a suitable mass extinction coefficient is available.
Calculate BSA protein concentration:
For more information about protein quantification, UV absorbance and extinction coefficients, see these authoritative resources:
For purified proteins, calculate from sequence using ProtParam (ExPASy) which counts Trp, Tyr, and Cys residues. Typical values: BSA = 0.7, IgG = 1.4, lysozyme = 2.6 (all in (mg/mL)⁻¹ cm⁻¹ at 280nm). For unknown proteins, use Bradford or BCA with BSA standard.
Spectrophotometers are accurate in absorbance range 0.1-1.0. Outside this range, measurements become nonlinear. If A > 1.0, dilute the sample. The dilution factor corrects back to original concentration. Most assays have optimal absorbance ranges for best accuracy.
A280: nucleic acids (absorb at 280nm), turbidity, reducing agents. Bradford: detergents (SDS, Triton), high salt, basic pH. BCA: reducing agents (DTT, β-ME), chelators (EDTA), lipids. Always include appropriate blanks with your buffer.
A280 for pure proteins (fastest, no reagents). Bradford for crude lysates (compatible with reducers). BCA for samples with detergents or when higher sensitivity needed. For unknown proteins, use Bradford or BCA with BSA standard curve.
For Bradford/BCA, prepare known BSA concentrations (0-2 mg/mL), measure absorbance, plot A vs concentration, fit a line/curve. Unknown protein absorbance is compared to this curve to determine concentration. Curves are method and plate-reader specific.
Bovine serum albumin (BSA) is cheap, pure, stable, and well-characterized. It's the universal protein standard. However, different proteins bind Bradford/BCA reagents differently, so BSA curves give approximate concentrations. For precision, use a purified version of your target protein as standard.
Yes. Microvolume (1mm path) uses only 1-2 µL sample. Standard cuvettes are 1 cm path. Formula still applies: shorter path = lower absorbance, so scale accordingly. Most spectrophotometers have cuvette adapters or built-in microvolume pedestal (e.g., NanoDrop).
A280: ±5% with known ε and pure protein. Bradford: ±10-15% due to protein-to-protein variation in dye binding. BCA: ±5-10%, more consistent across proteins. Always measure in triplicate and use freshly prepared standards for best accuracy.
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