Protein Concentration Calculator
Two ways to estimate protein concentration. (1) Beer–Lambert: enter your protein’s molar extinction coefficient and molecular weight to convert A280 into mg/mL. (2) A280 × 1 = mg/mL using the standard 1 A280 = 1 mg/mL assumption, which works for typical mixed-protein samples. The calculator also reports an A260/A280 purity note.
Calculator
Enter a reading and calculate to see the concentration
Concentration
c = A280 × dilution ÷ E(1 mg/mL) · c(M) = A280 × dilution ÷ ε
Beer–Lambert quantification: absorbance at 280 nm is proportional to concentration through the protein's extinction coefficient. In molar mode the result is also converted to mg/mL using the molecular weight.
About this calculation
What this is
Proteins absorb at 280 nm because of aromatic residues (tryptophan, tyrosine, and to a lesser extent cystine). For a purified protein with a known molar extinction coefficient, Beer–Lambert gives an accurate concentration; for a mixed or unknown sample, the heuristic 1 A280 = 1 mg/mL is a usable estimate.
Why it is used
Almost every downstream application — gel loading, dilution to a working stock, assay setup — needs a protein concentration. A280 is the fastest available measurement, but the assumption you make (1 mg/mL per A280 vs. Beer–Lambert) drives the accuracy of every result downstream.
Why this calculator exists
A280 readings are often reported in instrument software, but converting them into a usable mg/mL number is where mistakes happen — particularly the missing division by path length or the wrong extinction coefficient. The calculator does the unit bookkeeping and shows both the 1-A280 and Beer–Lambert results side by side.
Key assumptions
- The spectrophotometer was blanked with the same buffer the sample was diluted into.
- For the 1-A280 path, the protein mixture is roughly "average" (e.g. a crude lysate); for purified proteins use the calculated path.
- Path length is 1 cm, or the instrument has been corrected for a different path length.
Limitations
- The 1 A280 = 1 mg/mL assumption fails badly for samples rich in nucleic acids (use A260/A280 subtraction).
- It cannot detect proteins that lack aromatic residues or are present at sub-A280 noise-floor concentrations.
- It does not distinguish folded from denatured protein — the extinction coefficient assumes the folded environment.
What this calculates
- Concentration mg/mL
- Protein concentration in the original (undiluted) sample.
- Molar concentration µM
- Molar concentration — shown in ε + molecular-weight mode.
Frequently asked questions
How do I calculate protein concentration from an A280 reading?
The calculator applies the Beer–Lambert law to a blank-corrected A280: divide the absorbance by your extinction coefficient and multiply by the dilution factor. Provide either the mass coefficient E(1 mg/mL) to get mg/mL directly, or the molar coefficient ε together with the molecular weight to get both mg/mL and µM. Enter 1 as the dilution factor for an undiluted reading.
Should I use E(1 mg/mL) or the molar extinction coefficient?
Use whichever coefficient you actually have. E(1 mg/mL) is the absorbance of a 1 mg/mL solution in a 1 cm path, so it yields concentration in mg/mL on its own — BSA, for example, is about 1.4. The molar coefficient ε in M⁻¹·cm⁻¹ needs the molecular weight in g/mol as well, and it is the option that also reports the concentration in µM.
What does the A280 value actually measure in a protein sample?
Proteins absorb UV light at 280 nm mainly through their tryptophan, tyrosine, and cystine residues, so the absorbance scales with how much protein is in the path. That relationship is only fixed once you supply the coefficient specific to your protein, which is why the calculator asks for E(1 mg/mL) or ε. The upside is that the method needs only a few microlitres and leaves the sample usable for downstream work.
Why does the result show both mg/mL and µM, and when should I use each?
Both appear when you work in the molar mode, because the molecular weight lets the calculator convert between mass and amount. mg/mL is the practical unit for diluting a stock to a working concentration or loading a gel, while µM matters when molar stoichiometry drives the experiment, such as binding or enzyme assays. In E(1 mg/mL) mode only mg/mL is reported, since no molecular weight was entered.
What will make my A280 protein concentration wrong?
Nucleic acid contamination inflates A280 and pushes the calculated concentration high, so check the A260/A280 ratio of the sample and correct if needed. Buffer components that absorb near 280 nm, such as DTT at high concentration, imidazole, or Triton, bias the reading as well, so blank against the same buffer. The calculator assumes a 1 cm path length and a coefficient that matches your protein and its reducing state, and it applies no contamination correction of its own.
For research and educational use only. Not for clinical or diagnostic decisions. Always verify calculations independently before use in critical applications.