Protein
Concentration from A280
Convert absorbance into concentration for proteins and nucleic acids, derive the extinction coefficient from a sequence, and check the purity ratios.
Derive from a protein sequence
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Nucleic acids use the conventional factors: one absorbance unit equals 50 µg/ml for double-stranded DNA, 33 for single-stranded and 40 for RNA. A 260/230 ratio below 2 points to salt or phenol carry-over.
Formula
c = A ÷ (ε × l)
- A
- the measured absorbance
- ε
- molar extinction coefficient, per molar per centimetre
- l
- path length in centimetres
- nucleic acid
- one absorbance unit is 50 µg/ml for double-stranded DNA, 33 for single-stranded and 40 for RNA
Worked example
A protein of ε 43824 and 26900 Da reading 0.85
- A₂₈₀ = 0.85
- ε = 43824 M⁻¹cm⁻¹
- MW = 26900 Da
- l = 1 cm
- Molar concentration: 0.85 divided by 43824 is 1.94 × 10⁻⁵ M.
- To micromolar: multiply by 10⁶.
- Mass concentration: multiply the molar concentration by the molecular weight.
Answer 19.4 µM, which is 0.522 mg/ml
Common mistakes
- Forgetting the path length on a NanoDrop. It measures over 0.1 cm or even 0.03 cm and normalises to 1 cm itself; dividing again by the path is a tenfold error.
- Using the rule of thumb that one absorbance unit is one mg/ml for any protein. That only holds for an average aromatic composition and is badly wrong for a tryptophan-poor protein.
- Ignoring nucleic acid carry-over. DNA absorbs at 280 nm too and inflates the apparent protein concentration; check the 260/280 ratio.
- Leaving out the dilution factor when the sample was diluted before reading.
Frequently asked
What does the 260/280 ratio tell me?
Around 1.8 is expected for clean DNA and around 2.0 for RNA. A lower value points to protein or phenol. For a protein sample the logic reverses: a high ratio means nucleic acid contamination.
Why does 260/230 matter?
Below 2 usually means salt, phenol, carbohydrate or guanidine is still present. These interfere with downstream ligation and transfection even when the concentration itself was read correctly.
What if my protein has no tryptophan or tyrosine?
A280 will not work. Use BCA or Bradford, which respond to peptide bonds and basic residues, and fit the standard curve with the standard curve tool.