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Protein Concentration Calculator (A280, A205)

Calculate a protein or peptide concentration from A280 with an extinction coefficient or A 0.1%, from A205, or by Warburg-Christian; convert between mg/mL and micromolar.

Formula

c=A280×DFε ℓc = \dfrac{A_{280}\times\mathrm{DF}}{\varepsilon\,\ell}
c (mg/mL)=A280×DFA0.1% ℓc\,(\mathrm{mg/mL}) = \dfrac{A_{280}\times\mathrm{DF}}{A^{0.1\%}\,\ell}
c (mg/mL)=(1.55 A280−0.76 A260)×DFc\,(\mathrm{mg/mL}) = (1.55\,A_{280} - 0.76\,A_{260})\times\mathrm{DF}
c (mg/mL)≈A205×DF31c\,(\mathrm{mg/mL}) \approx \dfrac{A_{205}\times\mathrm{DF}}{31}
ε\varepsilon
molar extinction coefficient at 280 nm
A0.1%A^{0.1\%}
absorbance of a 1 mg/mL solution
MW\mathrm{MW}
molecular weight, for converting mass to moles

How it works

The most direct way to measure a pure protein is by its absorbance at 280 nm, converted to a concentration with an extinction coefficient calculated from its sequence or measured. When the extinction coefficient is unknown, the Warburg-Christian formula corrects A280 for nucleic acid using A260, and the absorbance of the peptide bond near 205 nm (about 31 absorbance units for 1 mg/mL) serves for peptides and proteins with few aromatic residues.

Whichever the method, the result can be converted between mg/mL and micromolar with the molecular weight. All of these are estimates of total protein in a clean sample; for a crude extract a colorimetric assay with a standard curve is usually better.

Worked example

A protein with ε₂₈₀ = 43,824 M⁻¹ cm⁻¹ and a molecular weight of 66,000 g/mol reads A280 = 0.65 in a 1 cm cuvette.

  1. c = 0.65 / (43,824 × 1) = 1.483 × 10⁻⁵ M = 14.83 µM.
  2. In mass terms, 14.83 µM × 66,000 g/mol = 0.9789 mg/mL.

0.979 mg/mL, which is 14.8 µM.

These are the values the calculator opens with, so you can check its output against this example.

Assumptions

  • The extinction coefficient or A 0.1% is that of the protein being measured, in the buffer used.
  • The reading is blank-corrected, within the linear range (about 0.1 to 1.0), with no turbidity.
  • For Warburg-Christian and A205, an average mixture of amino acids, which varies between proteins.

Common mistakes

  • Using BSA's extinction coefficient for another protein. The value is specific to the sequence.
  • Forgetting the dilution factor.
  • Measuring at 280 nm in the presence of nucleic acid, detergents or a buffer that absorbs there.

Related tools

Protein Extinction Coefficient Calculatorε280 and A 0.1% from sequence, with optional concentrationOpen calculatorBradford Protein Assay CalculatorProtein concentration from a Bradford standard curveOpen calculatorBCA Protein Assay CalculatorProtein concentration from a BCA standard curveOpen calculatorConcentration Unit ConverterMolar, mass per volume, mg/dL and % w/vOpen calculator

Related equipment

Service documentation, failure modes and parts for the instruments this calculation is used with.