Protein Concentration Calculator
Calculate protein concentration from A280 absorbance using the Beer-Lambert law. Free spectrophotometry calculator with BSA, IgG, Lysozyme and custom protein presets.
ε = 43,824 M⁻¹ cm⁻¹, MW = 66,463 g/mol
About This Calculator
The Protein Concentration Calculator determines the concentration of protein in your sample using UV absorbance readings from a spectrophotometer at 280 nm (A280). It is an essential tool for molecular biologists, biochemists, and laboratory researchers who need to quantify protein samples before downstream applications such as enzyme assays, electrophoresis, crystallography, or biophysical characterization.
The calculator uses the Beer-Lambert law (A = ε × c × l) adapted for protein quantification. The fundamental formula is: Concentration (mg/mL) = (A280 / (ε × b)) × m × n, where A280 is the absorbance at 280 nm, ε is the molar extinction coefficient (M⁻¹ cm⁻¹), b is the cuvette pathlength (cm), m is the molecular weight (g/mol), and n is the dilution factor. The calculator provides results in three units: mg/mL, µg/mL, and µM (micromolar), giving you flexibility for different laboratory protocols.
For accurate results, ensure your spectrophotometer is properly blanked with the appropriate buffer solution. The A280 reading should fall within the linear range of the instrument (typically 0.1–2.0 OD). If your reading is above 2.0, dilute your sample and enter the dilution factor to obtain the original sample concentration. Protein concentration determined by A280 is most reliable for purified proteins with known extinction coefficients — for complex mixtures or unknown proteins, colorimetric assays like Bradford or BCA may be more appropriate.
Regional Notes
India: Indian research institutions and biotech laboratories use the same spectrophotometric methods standardized globally. The Beer-Lambert law and extinction coefficients used here are universally accepted across Indian universities, CSIR labs, and biotech companies.
United States: US laboratories follow the same molecular biology and biochemistry standards. The A280 method is widely used in academic research, pharmaceutical development, and clinical labs. The BSA extinction coefficient of 43,824 M⁻¹ cm⁻¹ is referenced in standard protocols from vendors like Thermo Fisher and Bio-Rad.
United Kingdom: UK research institutions and MRC/BBSRC-funded laboratories adhere to the same protein quantification standards. The formula and constants in this calculator align with protocols from the Biochemical Society and the Royal Society of Chemistry.
Frequently Asked Questions
How do you calculate protein concentration from absorbance?
Protein concentration is calculated using the Beer-Lambert law: concentration (mg/mL) = (A / (ε × b)) × m × n, where A is absorbance at 280nm, ε is the extinction coefficient, b is cuvette pathlength, m is molecular weight, and n is the dilution factor.
What is a good A280 absorbance for protein quantification?
For reliable protein quantification, the A280 absorbance reading should be between 0.1 and 2.0 OD units. Readings below 0.1 have poor signal-to-noise ratio, while readings above 2.0 exceed the linear range of most spectrophotometers and require dilution.
What extinction coefficient should I use for BSA?
BSA (Bovine Serum Albumin) has an extinction coefficient of 43,824 M⁻¹ cm⁻¹ at 280 nm and a molecular weight of 66,463 g/mol. These values are widely accepted in molecular biology protocols and are pre-loaded in this calculator.
Why is 280 nm used for protein concentration measurement?
Proteins absorb UV light at 280 nm primarily due to the aromatic amino acids tryptophan and tyrosine. This absorption is proportional to protein concentration, making A280 measurement a quick, non-destructive method for protein quantification without the need for chemical reagents.
How does the Beer-Lambert law work for protein concentration?
The Beer-Lambert law (A = ε × c × l) states that absorbance is proportional to concentration and pathlength. For protein quantification, this is rearranged to c = A / (ε × l), giving concentration in molar units. Multiplying by molecular weight and dilution factor converts to mg/mL.
What is the difference between A280 and Bradford protein assay?
A280 measurement is a direct spectrophotometric method that requires no reagents and is non-destructive, but it requires a pure protein with a known extinction coefficient. The Bradford assay uses Coomassie dye and is more sensitive but destructive, and works with unknown proteins by comparing to a BSA standard curve.
How do I convert protein concentration from mg/mL to µM?
To convert mg/mL to µM, divide the concentration in mg/mL by the molecular weight (in g/mol), then multiply by 1,000,000. For example, 10 mg/mL of BSA (66,463 g/mol) equals approximately 150 µM.
Can I use this calculator for unknown proteins?
Yes, select the 'Custom protein' option and enter the extinction coefficient and molecular weight for your specific protein. These values are typically provided in the product documentation from commercial suppliers or can be calculated from the amino acid sequence using bioinformatics tools.