Protein Concentration (A280) Calculator
This calculator determines protein concentration from the ultraviolet absorbance at 280 nm using the Beer-Lambert law: c = A280 / (epsilon x l), where epsilon is the extinction coefficient in units of mL/(mg x cm), l is path length in cm, and c is concentration in mg/mL. The extinction coefficient is protein-specific: for many antibodies (IgG) it is approximately 1.4 mL/(mg x cm), for BSA it is approximately 0.667 mL/(mg x cm). If you know the molar mass and molar extinction coefficient, this calculator can also convert to mg/mL. Dilution factors are applied to report the concentration in the original undiluted sample.
Beer-Lambert protein formula
c (mg/mL) = A280 / (epsilon x l) x dilution factor
where epsilon = extinction coefficient (mL/mg/cm), l = path length (cm)
This is the simplified Beer-Lambert law using the specific extinction coefficient (also called E(0.1%)) for mass concentration, avoiding the need for molar mass.
Common extinction coefficients
- Human IgG antibody: approximately 1.35-1.40 mL/(mg x cm).
- BSA (bovine serum albumin): approximately 0.667 mL/(mg x cm).
- Lysozyme: approximately 2.64 mL/(mg x cm).
- GFP: approximately 2.28 mL/(mg x cm).
- Use ExPASy ProtParam (web.expasy.org) to compute the theoretical value from your protein sequence.
Frequently asked questions
Why is 280 nm used for protein concentration?
Tryptophan and tyrosine residues in proteins absorb ultraviolet light at 280 nm. Because most proteins contain at least some of these amino acids, absorbance at 280 nm provides a quick, non-destructive concentration estimate without reagents.
What is the Beer-Lambert law?
The Beer-Lambert law states that A = epsilon x c x l, where A is absorbance, epsilon is the molar extinction coefficient (L/mol/cm), c is molar concentration (mol/L), and l is path length (cm). Rearranged: c = A / (epsilon x l).
What extinction coefficient should I use?
The extinction coefficient is protein-specific and depends on its tryptophan, tyrosine, and disulfide bond content. For IgG antibodies a common value is about 1.4 (A280 per mg/mL per cm). ProtParam (ExPASy) can calculate the theoretical value from sequence. This calculator accepts any value you enter.
What is path length and what is the standard value?
Path length is the distance UV light travels through the sample, typically 1 cm in a standard cuvette. Microvolume spectrophotometers (e.g., NanoDrop) adjust for a very short path length (typically 0.1 mm or 1 mm) and report in equivalent 1 cm values automatically.
What factors interfere with A280 protein measurement?
Nucleic acid contamination (which also absorbs near 280 nm), detergents, reducing agents (DTT, beta-mercaptoethanol), and imidazole can all interfere. A correction using A260 and A280 together (Warburg-Christian method) can correct for nucleic acid contamination.
Official sources
- NCBI Bookshelf: Protein Quantification Methods.
- NIST: Reference absorption spectra and Beer-Lambert law.
Reviewed by the CalculatorHub team, edited by James Graham, 15 June 2026. See our methodology.