The formula
mg/mL = A280 × dilution ÷ (Abs 0.1% × path length in cm)
Abs 0.1% is the absorbance of a 1 mg/mL solution in a 1 cm path. It is the E1% value divided by 10, or the molar extinction coefficient divided by the molecular weight. Everything depends on using the right value for your protein, because proteins with more tryptophan and tyrosine absorb more strongly.
Antibodies
For IgG the usual E1% is 13.7, so an A280 of 1.37 in a 1 cm path is 1 mg/mL. Published values for different species and subclasses run from about 13.5 to 14. Measure the antibody before conjugation or line coating: once it is bound to gold or latex the particles absorb as well, and A280 no longer tells you the protein concentration.
Worked example
An IgG diluted 1:2 reads A280 = 0.685, with A320 = 0.010 for scatter. Corrected A280 is 0.675, so the concentration is 0.675 × 2 ÷ 1.37 = 0.9854 mg/mL, about 6.57 µM for a 150 kDa antibody.
Extinction coefficient from the sequence
Paste the amino acid sequence and the extinction coefficient is worked out the same way as ExPASy ProtParam: 5500 for each tryptophan, 1490 for each tyrosine and 125 for each cystine (a pair of cysteines joined by a disulfide bond), in M⁻¹ cm⁻¹. The molecular weight comes from the sequence too. Choose whether the cysteines are paired; secreted proteins and antibodies usually have their cysteines in disulfide bonds. A protein with no tryptophan or tyrosine barely absorbs at 280 nm, so use a colorimetric assay instead.
Purity and scatter
Nucleic acids absorb at 280 nm too, so contamination makes the protein reading high. An A260/A280 ratio of about 0.57 is typical of pure protein, and above 0.6 suggests nucleic acid. Aggregates and dust scatter light at every wavelength; reading at 320 or 340 nm, where proteins don't absorb, and subtracting it corrects for that.
Readings are most reliable between about 0.1 and 1.5. Plate readers usually have a path length well under 1 cm, so enter it, or use a reader that corrects to 1 cm.
This calculator is checked against independent calculations before every release. How the calculators are checked.
References
- Pace CN, Vajdos F, Fee L, Grimsley G, Gray T (1995). How to measure and predict the molar absorption coefficient of a protein. Protein Science 4: 2411–2423.