A280 protein concentration calculator
Maintained by the Talindrew team · Last updated
Protein concentration from absorbance at 280 nm follows Beer–Lambert: mg/ml = A280 × MW / ε, with ε the molar extinction coefficient (M⁻¹cm⁻¹) and a 1 cm path. For BSA (ε 43,824, MW 66,463) an A280 of 1.0 is 1.52 mg/ml. Tryptophan, tyrosine and cystines are what absorb: ε is protein-specific.
Result
Beer–Lambert: mg/ml = A280 × MW / ε; µM = A280/ε × 10⁶. Defaults are BSA (ε 43,824 M⁻¹cm⁻¹, MW 66,463 Da per ExPASy ProtParam). Get ε for your protein from its sequence. Talindrew's protein tools compute it by the Pace 1995 method.
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What it looks like
Formula
c (mg/ml) = A280 × MW / ε · c (µM) = A280 / ε × 10⁶
Source: Beer–Lambert law; ε predicted from sequence per Pace et al., Protein Sci 1995
Worked example
Given: BSA solution, A280 = 1.0 (ε 43,824 M⁻¹cm⁻¹, MW 66,463 Da per ProtParam)
- 1.Molar concentration: 1.0 / 43,824 = 22.8 µM.
- 2.Mass concentration: 1.0 × 66,463 / 43,824 = 1.52 mg/ml.
A280 1.0 of BSA = 1.52 mg/ml (22.8 µM)
How the calculation flows
Units & constants
| What absorbs at 280 nm | Trp (ε ~5,500) ≫ Tyr (~1,490) > cystine (~125) per residue |
|---|---|
| ε prediction | Pace 1995 method from sequence (ProtParam; Talindrew protein tools) |
| BSA reference | ε 43,824 M⁻¹cm⁻¹, MW 66,463 Da |
| Lysozyme reference | ε 37,970 M⁻¹cm⁻¹, MW 14,313 Da |
| Reliable A280 range | ~0.1–1.0; dilute and multiply back |
| Caveat | Nucleic acid contamination inflates A280 (check A260/A280) |
| Price | Free |
Where do you get ε for your protein?
Compute it from the sequence: the Pace 1995 method sums contributions of tryptophans, tyrosines and disulfide-bonded cystines, and is what ExPASy ProtParam and Talindrew's protein properties report. Use the value matching your protein's state, reduced (free cysteines) or with disulfides formed, they differ by 125 M⁻¹cm⁻¹ per cystine.
When is A280 the wrong method?
A280 fails predictably in a few cases:
- Proteins with no Trp/Tyr (some small peptides): ε is tiny and the reading is noise, so use BCA or Bradford.
- Crude lysates: everything absorbs; A280 reads total aromatic content, not your protein.
- Detergent- or nucleotide-containing buffers: background absorbance shifts, so always blank with the exact buffer.
- Nucleic acid contamination: DNA absorbs strongly at 280 too; an A260/A280 above ~0.7 for a pure protein flags trouble.
Frequently asked questions
A rough average (ε/MW ≈ 1 (mg/ml)⁻¹cm⁻¹) that happens to be nearly right for antibodies (IgG: 1.35–1.4) and wrong by up to two-fold for many proteins. With a computed ε there is no reason to use it.
Yes: enter the dilution factor and the result is scaled back to the stock concentration, the same convention as the OD260 nucleic-acid calculator.
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