A280 (Absorbance at 280 nm) Calculators

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A280 refers to the absorbance of a solution at 280 nm ultraviolet light, used to quantify protein concentration. Aromatic amino acids tryptophan (W) and tyrosine (Y), and to a lesser extent cysteine disulfide bonds, absorb UV light at 280 nm. The protein concentration can be calculated from A280 using the Beer-Lambert law: C (mg/mL) = A280 / (ε × path length), where ε is the molar extinction coefficient (M⁻¹cm⁻¹) or the specific extinction coefficient E280 (mL/mg/cm). The NanoDrop spectrophotometer is the most common instrument for rapid A280 measurements requiring only 1–2 μL of sample.

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Beer-Lambert Law for A280

A₂₈₀ = ε × c × l

ε = extinction coefficient; c = molar concentration (M); l = path length (cm, usually 1 cm). For protein concentration in mg/mL: C = A₂₈₀ / (E₁% × l) or C = A₂₈₀ × MW / (ε × l). E₁% = specific extinction coefficient (A₂₈₀ at 1% w/v in 1 cm path); varies by protein composition.

Common Protein A280 Values (E₁%, 1 mg/mL in 1 cm)

  • IgG antibody: E₁% ≈ 1.35 A280/(mg/mL)
  • BSA: E₁% ≈ 0.66
  • Streptavidin: E₁% ≈ 3.40

Calculated from sequence: use ExPASy ProtParam tool (input amino acid sequence → calculates ε from Trp, Tyr, Cys content).

A260/A280 Ratio (Nucleic Acid Contamination)

Pure protein: A260/A280 ≈ 0.57. If ratio > 0.7: nucleic acid contamination (DNA/RNA absorb strongly at 260 nm). To quantify both: pure dsDNA A260/A280 ≈ 1.8; pure ssRNA ≈ 2.0.

Limitations of A280

  • Proteins without Trp/Tyr: near zero A280 → use alternative methods (BCA, Bradford)
  • Aggregates scatter light → overestimate A280
  • Phenol, EDTA, imidazole, detergents absorb at 280 nm → background interference
  • NanoDrop: quick and easy; A280 not as accurate as BCA/Bradford for very low concentrations

Glossary

A280
Absorbance at 280 nm used to quantify protein concentration; aromatic residues Trp and Tyr absorb UV at 280 nm; C = A₂₈₀/(E₁% × path length); measured by NanoDrop or UV-Vis spectrophotometer.
Molar Extinction Coefficient (ε)
The absorbance per unit molar concentration and path length (M⁻¹cm⁻¹) at a given wavelength; calculated from Trp+Tyr+Cys content using ExPASy ProtParam; used in Beer-Lambert: A = ε × c × l.
A260/A280 Ratio
A purity indicator: pure dsDNA ≈ 1.8–2.0; pure ssRNA ≈ 2.0; pure protein ≈ 0.57; ratio below expected indicates contamination with protein (for nucleic acids) or nucleic acids (for proteins).

Frequently Asked Questions

A280 is the absorbance of a protein solution at 280 nm UV wavelength. Tryptophan (W) and tyrosine (Y) residues in proteins strongly absorb at 280 nm; disulfide bonds (cysteine pairs) contribute weakly. Protein concentration is calculated using Beer-Lambert law: A = ε × c × l, rearranged to c = A/(ε × l). For a known protein: use the known molar extinction coefficient ε (M⁻¹cm⁻¹) or specific extinction coefficient E₁% (absorbance of 1 mg/mL in 1 cm path) calculated from the amino acid sequence using ExPASy ProtParam. For unknown proteins: use an average extinction coefficient (e.g., E₁% ≈ 1.0 for typical globular proteins) as a rough approximation.

Method 1: Using specific extinction coefficient E₁%: C (mg/mL) = A₂₈₀ / E₁%. Example: A₂₈₀ = 0.826; E₁% = 1.35 (IgG): C = 0.826/1.35 = 0.61 mg/mL. Method 2: Using molar extinction coefficient ε: C (M) = A₂₈₀ / (ε × path length cm). Then convert to mg/mL: C (mg/mL) = C (M) × MW (g/mol) / 1000. Method 3: NanoDrop 2000 direct reading: enter protein MW and extinction coefficient; instrument calculates mg/mL automatically. For unknown proteins without sequence data: use Bradford or BCA assay with a BSA standard curve instead of A280.

A260/A280 ratio indicates sample purity: For protein: pure protein has A260/A280 ≈ 0.57 (low 260 absorbance because proteins lack nucleobases). If A260/A280 > 0.7: nucleic acid contamination — RNA/DNA absorb strongly at 260 nm. Contaminant causes overestimation of protein at A280. For nucleic acids (NanoDrop): pure dsDNA: A260/A280 ≈ 1.8–2.0; if ratio < 1.8: protein contamination or phenol/other contaminants. Pure ssRNA: A260/A280 ≈ 2.0. Low A260/A280 for DNA/RNA usually indicates protein contamination or phenol carryover from extraction. Both ratios are contamination indicators — a ratio in the expected range doesn't guarantee purity (some contaminants don't affect these ratios).

Limitations of A280: (1) Proteins without Trp or Tyr (or with very few): near-zero extinction coefficient → A280 cannot quantify them; examples: collagen, glycine-rich proteins. (2) Light scattering: aggregated proteins or turbid solutions scatter light at 280 nm → falsely elevated A280. (3) Chemical interference: EDTA (absorbs 280 nm at high concentrations); imidazole (absorbs 280 nm — problem with His-tag purification elution buffers); phenol (from RNA extraction); some detergents (SDS absorbs around 260–280 nm). (4) Limited accuracy: A280 is less precise than colorimetric assays (BCA, Bradford) at low concentrations. Use A280 for: quick quality control of purified proteins; monitoring column fractions; high-concentration samples where linearity is maintained. Use BCA/Bradford for: absolute quantification; complex mixtures; low concentrations.