What protein concentration does this absorbance mean?

A280 protein concentration calculator (Beer-Lambert)

Converts absorbance at 280 nm (A280) into the concentration of an antibody or other protein in mg/mL, correcting for any dilution before measurement and for the cell's path length.

When to use itUse it for a quick spectrophotometer check of in-process samples or drug substance.

Prefilled with an example: an antibody with an extinction coefficient of 1.4 that reads 0.8. Change any value and the result updates.

Measured absorbance (A280)

Abs

Absorbance at 280 nm. Dilute so the reading falls between 0.1 and 1.0. If you corrected for turbidity, enter the value after subtracting the reading at 320 to 340 nm.

Extinction coefficient (absorbance of 1 mg/mL in 1 cm, A0.1%)

mL·mg⁻¹·cm⁻¹

Specific to each protein; about 1.4 to 1.5 for IgG antibodies. Use a value calculated from the amino acid sequence or a measured one. If you only have the molar extinction coefficient ε, divide it by the molecular weight in Da. If you use the value per 10 mg/mL (E1%, about 14 for IgG), divide it by 10.

Path length

1 cm for a standard cuvette. For microvolume instruments, enter the path length the instrument actually uses (for example, 0.1 cm).

Dilution before measuring

Enter 10 if you diluted 10 times, or 1 if you did not dilute.

Protein concentration0.5714 mg/mL

The protein concentration of this sample is about 0.5714 mg/mL. The absorbance is within 0.1 to 1.0, so the reading is trustworthy as far as the measurement goes.

Guide to the measured absorbance

The row that matches your result is marked.

Absorbance 0.1 to 1.0 (as measured, after dilution)Your resultLinear rangeThe working range given in our related article (roughly 0.1 to 1.0), where absorbance and concentration are most likely to be proportional and the number is most trustworthy. It is a guide to measurement, not a product specification.
Absorbance below 0.1Too low (possibly overdiluted)Noise and measurement error weigh more. Consider remeasuring with less dilution. This is a guide, not a pass/fail decision.
Absorbance above 1.0Too high (possibly underdiluted)Absorbance starts to level off and the concentration tends to be underestimated. Dilute further to bring it within 0.1 to 1.0. This is a guide, not a pass/fail decision.
Turbidity, aggregates, excipients or other proteins likely presentA280 alone may read highLight scattering from turbidity, and absorbance by anything other than the antibody, are picked up too. Combine with a correction at 320 to 340 nm or an antibody-specific method such as Protein A HPLC (see the related article). Content decisions for drug substance or drug product follow the product specification and the pharmacopoeia, not this calculator.

How it is calculated

Concentration = absorbance ÷ (extinction coefficient × path length) × dilution factor. The extinction coefficient is the absorbance of a 1 mg/mL solution in a 1 cm cell, so dividing the absorbance by it gives mg/mL. Dividing by the path length and multiplying by the dilution factor takes you back to the original sample.

C [mg/mL] = A280 ÷ (extinction coefficient A0.1% × path length) × dilution factor

Source: By the Beer-Lambert law, concentration = A280 ÷ (extinction coefficient × path length). The extinction coefficient can be estimated from the amino acid sequence, for example by the method of Pace CN et al. (1995).

With your numbers

concentration = A280 ÷ (extinction coefficient A0.1% × path length) × dilution = 0.8 ÷ (1.4 × 1) × 1 = 0.5714 mg/mL

Limits of this calculation

The extinction coefficient is specific to each antibody, and the representative IgG value of 1.4 to 1.5 mL/mg/cm in our related article is only typical. As a rule, use a value calculated from the amino acid sequence or confirmed by measurement; the default of 1.4 is for estimates.
The result is the total of all protein that absorbs at 280 nm. Proteins other than the antibody (host cell proteins, medium components) and excipients can push it up. To measure the antibody alone, combine with a method such as Protein A HPLC (see the related article).
Scattering, turbidity and aggregation affect the reading. Without a correction such as subtracting the value near 320 to 340 nm, the concentration tends to be overestimated.
The calculation assumes the absorbance is measured within the linear range (roughly 0.1 to 1.0). If it falls outside, adjust the dilution.
This calculator is an estimate from a standard equation and does not replace pass/fail testing of content or potency for drug substance or drug product. Final decisions follow the product specification based on ICH Q6B and similar guidelines, and the pharmacopoeia.
Converting from the molar extinction coefficient ε requires the molecular weight (extinction coefficient per 1 mg/mL = ε ÷ molecular weight). Antibodies are about 145,000 to 150,000 Da, but this varies by product, so use the value for yours.

Primary sources

Every calculator gives an estimate for learning, built on published information and primary sources. Real decisions follow the applicable guidance and expert judgment.