Protein concentration: A280, extinction coefficient and kDa ↔ molar
Everything you need between a spectrophotometer reading and a molar concentration — including molecular weight and ε₂₈₀ computed straight from the sequence.
Weight ↔ molar quantity kDa
Convert a protein mass to moles and back. Enter the mass or the amount — whichever you know.
Concentration from A₂₈₀ Beer–Lambert
Works with a molar ε (M⁻¹cm⁻¹), a 0.1 % coefficient (A of a 1 mg/mL solution) or the generic “A₂₈₀ = 1 ≈ 1 mg/mL” assumption.
Molecular weight and ε₂₈₀ from sequence
Paste a one-letter amino-acid sequence (FASTA headers and whitespace are ignored).
| Property | Value |
|---|
Protein mg/mL ↔ µM
The same conversion as on the molarity page, pre-set for protein-scale numbers.
Choosing a quantitation method
| Method | Range | Strengths | Watch out for |
|---|---|---|---|
| A₂₈₀ | 0.05–5 mg/mL | Non-destructive, instant, sample recoverable | Needs W/Y; nucleic acid and scattering inflate it |
| Bradford | 1–1500 µg/mL | Fast, tolerant of reducing agents | Strongly protein-to-protein variable; detergents interfere |
| BCA | 20–2000 µg/mL | Detergent-compatible, low protein-to-protein variation | Reducing agents and chelators interfere; needs 37 °C |
| Lowry | 5–100 µg/mL | Sensitive, long-established | Many interfering substances; multi-step |
| Amino-acid analysis | any | Absolute, the reference method | Destructive, slow, expensive |
Interpreting A260/A280
Pure protein has an A260/A280 ratio near 0.57; pure DNA is about 1.8. The Warburg–Christian approximation corrects for modest nucleic-acid contamination:
protein (mg/mL) ≈ 1.55 × A₂₈₀ − 0.76 × A₂₆₀
It is only an approximation — for anything quantitative, remove the nucleic acid rather than correcting for it.
Why some proteins cannot be measured at 280 nm
The A₂₈₀ signal comes almost entirely from tryptophan and tyrosine. A protein with no W and no Y — some small peptides, collagen fragments, many antimicrobial peptides — has an ε₂₈₀ of nearly zero and is invisible at 280 nm. Use a colourimetric assay, or measure the peptide bond directly at 205–214 nm, where ε is roughly 30–35 (mg/mL)⁻¹cm⁻¹ at 205 nm.
Reference values
| Protein | MW (kDa) | ε₂₈₀ (M⁻¹cm⁻¹) | A₂₈₀ at 1 mg/mL |
|---|---|---|---|
| Bovine serum albumin (mature) | 66.43 | 43 824 | 0.660 |
| Lysozyme (hen egg white) | 14.31 | 38 940 | 2.72 |
| IgG (typical) | 150 | 210 000 | 1.40 |
| GFP (avGFP) | 26.9 | 21 890 | 0.814 |
| Ubiquitin | 8.56 | 1 490 | 0.174 |
| Chymotrypsinogen A | 25.7 | 50 585 | 1.97 |
Frequently asked questions
How do I calculate protein concentration from A280?
Beer–Lambert: c = A₂₈₀ / (ε × l). With a molar extinction coefficient ε in M⁻¹cm⁻¹ and a 1 cm path you get molar concentration directly; with a 0.1% (1 g/L) coefficient you get mg/mL.
How is the extinction coefficient at 280 nm estimated?
From tryptophan, tyrosine and cystine content (Pace et al., 1995): ε = nW×5500 + nY×1490 + nCystine×125 M⁻¹cm⁻¹. Free cysteines in a reduced protein contribute essentially nothing.
How do I convert mg/mL of protein to µM?
Divide the concentration in g/L by the molecular weight in g/mol. For a 66.5 kDa protein at 1 mg/mL: 1 / 66 500 = 1.504 × 10⁻⁵ M = 15.04 µM.
Why does my A280 reading look too high?
Nucleic-acid contamination absorbs strongly at 260 nm and bleeds into 280 nm; check the A260/A280 ratio (pure protein ≈ 0.57). Light scattering from aggregates raises the whole spectrum — subtract the A330 baseline.