Protein A280 Concentration Calculator

Calculate protein concentration from A280 using Beer-Lambert law and extinction coefficient.

Absorbance at 280nm (A280)

Molar extinction coefficient ε280 (M⁻¹cm⁻¹) (M⁻¹cm⁻¹)

BSA=43824, IgG~210000

Molecular weight (kDa) (kDa)

BSA=66.5kDa

Path length l (cm) (cm)

Please check your inputs and try again.

Two Ways to Report Concentration

Beer–Lambert gives molar concentration directly. Converting to mg/mL requires the molecular weight, and mixing up the two units is the commonest error on this calculation.

c (M) = A / (ε × l) c (mg/mL) = c (M) × MW (kDa)

Route	When to use
Molar, from ε and MW	Sequence known — most accurate
A280 × 1 mg/mL coefficient	Sequence known, coefficient tabulated
A280 ≈ 1 for 1 mg/mL IgG	Antibodies — rule of thumb

The IgG shortcut works because antibodies have relatively conserved aromatic content. It should not be applied to other proteins, where extinction coefficients vary by an order of magnitude.

What Inflates A280

Interference	Effect	Detection
Nucleic acid	Strongly inflates	A260/A280 above 0.6
Aggregates and turbidity	Scattering raises baseline	Non-zero A320
Imidazole, DTT at high concentration	Absorb near 280	Blank against buffer
Haem, flavin, other cofactors	Absorb at 280	Use corrected coefficient

Pure protein gives an A260/A280 ratio near 0.57; pure nucleic acid gives about 1.8. Because nucleic acids have far higher extinction coefficients, even 5% DNA contamination inflates apparent protein concentration substantially.

Scattering from aggregates raises absorbance across the spectrum. Since protein does not absorb at 320 nm, any reading there indicates turbidity and should be subtracted from A280 before calculating.

When A280 Cannot Be Used

Proteins lacking tryptophan have low extinction coefficients, and those lacking both tryptophan and tyrosine are effectively invisible. Collagen and many structural proteins fall into this category.

The alternatives have their own biases: Bradford responds mainly to arginine and aromatic residues, so it varies severalfold between proteins, while BCA is more uniform but interfered with by reducing agents. A280 remains preferred where it works because it is non-destructive and requires no standard curve.

Worked Examples

Example 1: BSA: A280=0.85, ε=43824, MW=66.5

c=0.85/43824=1.94×10⁻⁵ M

Result: c=1.29 mg/mL

Standard for Bradford/BCA calibration

Example 2: IgG antibody: A280=1.2, ε=210000

c=1.2/210000=5.7×10⁻⁶ M

Result: c=0.86 mg/mL at MW=150kDa

Typical IgG working concentration for experiments

Example 3: Detecting contamination

A260/A280 = 1.1 instead of 0.57

Result: Substantial nucleic acid present

Apparent protein concentration is inflated. Nuclease treatment or further purification is needed before A280 can be trusted.

Example 4: When A280 fails

Protein with no tryptophan or tyrosine

Result: Effectively invisible at 280 nm

Use BCA or Bradford, or measure at 205 nm where the peptide backbone absorbs regardless of side chain composition.

Common Mistakes

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Confusing molar with mass concentration

Dividing absorbance by ε gives molarity. Multiplying by molecular weight in kDa converts to mg/mL.

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Ignoring nucleic acid contamination

Nucleic acids absorb strongly at 280 and have much higher extinction coefficients. Check that A260/A280 is near 0.57.

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Not subtracting a scattering baseline

Aggregates scatter across the spectrum. Protein does not absorb at 320 nm, so any signal there should be subtracted from A280.

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Applying the IgG rule of thumb to other proteins

A280 of 1 for 1 mg/mL holds only for antibodies. Extinction coefficients vary by an order of magnitude between proteins.

Frequently Asked Questions

ε280 sources?

Pace/Gill & von Hippel equation: ε=5500×W + 1490×Y + 125×C (disulfides). ExPASy ProtParam from sequence. Most accurate: measure directly using amino acid analysis. For antibodies: use extinction coefficient tables from literature.

A280 vs BCA/Bradford?

A280: non-destructive, fast, no reagents needed. Limitation: interfering compounds (nucleic acids, HEPES, Tris buffer at high conc). BCA/Bradford: measures total protein regardless of composition. Choose based on sample purity and available standards.

How do I convert molar concentration to mg/mL?

Multiply by molecular weight in kDa. Absorbance divided by the extinction coefficient gives molarity first.

What A260/A280 ratio indicates pure protein?

About 0.57. Values approaching 1.8 indicate substantial nucleic acid contamination, which inflates apparent protein concentration.

Why subtract A320?

Protein does not absorb at 320 nm, so any reading there reflects light scattering from aggregates and should be removed from A280.

When should I use Bradford or BCA instead?

When the protein lacks aromatic residues, or when the sample contains interfering absorbers. Note Bradford responds mainly to arginine and varies severalfold between proteins.