Protein Concentration Calculator | A280 to mg/mL
Convert an A280 absorbance reading into protein concentration in mg/mL using the Beer-Lambert law, with coefficients for BSA, IgG, lysozyme, and ovalbumin.
Protein Concentration Calculator
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Protein concentration calculator
A protein concentration calculator converts a light-absorbance reading from a spectrophotometer into the amount of protein dissolved in a solution. The result is usually given in milligrams per milliliter (mg/mL) or micrograms per milliliter (μg/mL). The calculation is based on the Beer-Lambert law, a relationship in chemistry that links how much light a sample absorbs to how much of a substance it contains.
How protein concentration is measured
Most proteins absorb ultraviolet light strongly at a wavelength of 280 nanometers (nm). This absorption comes mainly from two amino acids, tryptophan and tyrosine, found in the protein's structure. A spectrophotometer shines light through the sample and measures how much of it the sample absorbs. This reading is called absorbance, or A280 when taken at 280 nm.
Measuring absorbance at 280 nm is fast and does not destroy the sample. That sets it apart from chemical methods such as the Bradford or BCA assay, described below. The method works best on a purified protein in solution, with an absorbance reading between about 0.1 and 1.0. Readings outside that range are less reliable, so a sample often needs to be diluted before measurement.
Protein concentration formula
The calculator rearranges the Beer-Lambert law to solve for concentration:
C = (A × DF) / (ε × l)
- C — protein concentration, in mg/mL
- A — absorbance at 280 nm (a plain number, with no unit)
- DF — dilution factor, how many times the original sample was diluted before it was measured
- ε — extinction coefficient, a constant specific to the protein, in L/(g·cm)
- l — path length, the distance light travels through the sample, in cm (usually 1 cm in a standard cuvette, a small glass or plastic container used in the spectrophotometer)
To convert the result to micrograms per milliliter, multiply the mg/mL value by 1,000. To find the total mass of protein in the sample, multiply the concentration by the sample's volume in milliliters.
Example calculation
A sample gives an absorbance reading of 0.5 at 280 nm. It was not diluted, so the dilution factor is 1, and it was measured in a standard 1 cm cuvette. The protein is bovine serum albumin (BSA), which has an extinction coefficient of 0.667 L/(g·cm).
C = (0.5 × 1) / (0.667 × 1) ≈ 0.75 mg/mL
In a 1 mL sample, that is about 0.75 mg of total protein, or 750 μg/mL.
Extinction coefficients for common proteins
Each protein absorbs light differently, because each has a different number of tryptophan and tyrosine residues. The extinction coefficient describes this for a specific protein at 280 nm, in units of L/(g·cm). Commonly used values include:
| Protein | Extinction coefficient ε (L/(g·cm)) |
|---|---|
| Bovine serum albumin (BSA) | 0.667 |
| Immunoglobulin G (IgG) | 1.38 |
| Lysozyme | 2.64 |
| Ovalbumin | 0.73 |
A calculator can also accept a custom extinction coefficient for a protein that is not on this list. For a protein with a known amino acid sequence, this value can be estimated with a formula published by Gill and von Hippel in 1989: ε₂₈₀ = (tryptophan residues × 5,500) + (tyrosine residues × 1,490) + (cystine residues × 125). That formula gives a result in M⁻¹cm⁻¹, a molar unit, which differs from the mass-based L/(g·cm) values above. Free tools such as ProtParam, hosted by the ExPASy server, calculate this value directly from a protein sequence.
Path length and dilution factor
The path length is the width of the cuvette that holds the sample, measured in centimeters. Standard cuvettes are 1 cm wide. Microvolume spectrophotometers, which measure very small samples, may use a much shorter path, such as 0.1 cm.
The dilution factor accounts for any dilution performed before measurement. If a concentrated sample would give an absorbance above 1.0, it is common to dilute it with buffer first. The dilution factor equals the total diluted volume divided by the original sample volume. For example, mixing 10 μL of sample with 990 μL of buffer gives a total volume of 1,000 μL, a dilution factor of 100.
Other ways to measure protein concentration
Absorbance at 280 nm is one of several common methods used in a laboratory.
- Bradford assay: uses a dye that changes color when it binds to protein. It works well on unpurified samples but the color change can vary between different proteins.
- BCA assay: relies on a color change caused by the protein's peptide bonds. It is compatible with many detergents but takes longer to run than a direct absorbance reading.
- Lowry method: an older method that is very sensitive but reacts poorly with some common buffer ingredients.
Each of these three methods destroys the sample. A direct A280 measurement does not, so the same sample can be recovered and used again.
Frequently asked questions
What is the ideal absorbance range for this method?
Between 0.1 and 1.0. Below 0.1, the reading is imprecise. Above 1.0, the relationship between absorbance and concentration may no longer be a straight line, so the sample should be diluted first.
Why measure at 280 nm instead of 260 nm?
Nucleic acids, such as DNA and RNA, absorb strongly at 260 nm. Proteins absorb more at 280 nm because of their tryptophan and tyrosine content. Measuring at 280 nm reduces interference from any nucleic acids present in the sample.
How is the extinction coefficient found for a protein that isn't listed?
For a protein with a known sequence, a tool such as ProtParam calculates it from the number of tryptophan, tyrosine, and cystine residues. It can also be measured directly, by comparing the sample's absorbance to a solution of known concentration.
Does this method work for a mixture of proteins?
It works best for a single, purified protein with a known extinction coefficient. A mixture of unknown proteins, such as a cell lysate, is usually measured with the Bradford or BCA assay instead, since those methods give a more consistent estimate of total protein.
Can detergents interfere with the reading?
Yes. Many detergents absorb light near 280 nm, which can distort the result. Using a blank sample that contains the same detergent concentration helps cancel out that background absorbance. Very high detergent concentrations may call for a detergent-compatible assay, such as BCA, instead.