Free Protein Concentration Calculator

Protein

Absorbance (A280)

Pathlength (cm)

Dilution Factor

Enter values to calculate

Protein Concentration Calculation Using the A280 Method

This protein concentration calculator is a free online tool that applies the Beer–Lambert law to determine the concentration of a protein from its UV absorbance at 280 nm. Often referred to as the A280 protein assay, this technique is rapid, non‑destructive, and requires no additional reagents, making it ideal for routine quantification of purified proteins.

The Beer–Lambert Relationship

The core equation is:

A = \varepsilon \cdot b \cdot C

where $A$ is the absorbance at 280 nm, $\varepsilon$ is the molar extinction coefficient (in $\text{M}^{-1}\text{cm}^{-1}$ ), $b$ is the pathlength of the cuvette (in cm), and $C$ is the molar concentration (M). To obtain the mass concentration in mg/mL, the formula is expanded with the molecular mass $m$ (g/mol) and the dilution factor $n$ :

C_{\text{mg/mL}} = \frac{A}{\varepsilon \cdot b} \times m \times n

Multiply the result by 1000 to get the concentration in µg/mL.

Parameters You Need to Provide

Five values must be supplied:

Absorbance ( $A$ ): The optical density at 280 nm, measured with a spectrophotometer. For best accuracy, keep readings in the 0.1–1.0 range.
Extinction coefficient ( $\varepsilon$ ): A protein‑specific constant that depends on the content of tryptophan, tyrosine, and cysteine. It can be found in the protein’s documentation or in the reference table below.
Pathlength ( $b$ ): Usually 1 cm for standard cuvettes. If you use a different cuvette, enter its internal width.
Molecular mass ( $m$ ): The molar mass of the protein (g/mol). Many common proteins have pre‑loaded values in the calculator; for others, you can look up the mass in published literature.
Dilution factor ( $n$ ): Set to 1 if you measured the undiluted stock. For a diluted sample, $n$ is the reciprocal of the dilution ratio (e.g., 2 for a 1:2 dilution, 10 for a 1:10 dilution).

Step‑by‑Step Example

Let’s determine the concentration of an Immunoglobin G (IgG) sample with the following data:

Extinction coefficient: 210,000 M⁻¹ cm⁻¹
Molecular mass: 150,000 g/mol
Measured absorbance: 0.55
Cuvette pathlength: 1 cm
Dilution factor: 5 (1:5 dilution)

Insert these into the formula:

C = \frac{0.55}{210,000 \times 1} \times 150,000 \times 5 = \frac{0.55}{210,000} \times 750,000 \approx 1.964\ \text{mg/mL}

Thus, the stock concentration is about 1.96 mg/mL (1960 µg/mL).

Reference Table of Extinction Coefficients and Molecular Masses

The following table lists the parameters for all proteins and amino acids pre‑programmed in the calculator. Data are sourced from BioSynthesis USA and von Hippel & Gill, measured at 280 nm.

Substance	Molecular Mass (g/mol)	Extinction Coefficient (M⁻¹ cm⁻¹)
Aldose	38,994	34.380
APC – Allophycocyanin	105,000	700,000
ATP	507.2	15,400
BSA – Bovine Serum Albumin	66,463	43,824
Cysteine	121.16	120
IgG – Immunoglobin G	150,000	210,000
Insulin	5,734	6,335
Lysozyme	14,000	37,901
PE – Phycoerythrin	240,000	1,960,000
Phenylalanine	165.19	200
RNAse A	13,700	9,440
Streptavidin	55,000	176,000
Tryptophan	204.23	5,500
Tyrosine	181.19	1,490

If the protein you are working with is not included in this list, the calculator offers a “Custom” option that allows you to manually enter the extinction coefficient and molecular mass.

This tool streamlines the A280 protein assay workflow, eliminating manual calculations and reducing the chance of arithmetic errors.

FAQ

1. How is protein concentration calculated from absorbance at 280 nm?

The calculation uses the formula C = (A / (ε·b)) × m × n, where A is the absorbance at 280 nm, ε is the extinction coefficient, b is the pathlength, m is the molecular mass in g/mol, and n is the dilution factor. The result is in mg/mL.

2. What extinction coefficient should I use for a protein not listed in the table?

If your protein is not in the reference table, select the “Custom” option in the calculator and enter its extinction coefficient and molecular mass manually. These values are typically available in the protein’s datasheet or published literature.

3. How does the dilution factor affect the concentration result?

The dilution factor (n) linearly scales the calculated concentration. For a 1:X dilution, enter X. If the undiluted stock is measured, set n = 1. The result reflects the concentration of the original sample before dilution.

4. Can I convert the protein concentration from mg/mL to µg/mL?

Yes, multiply the concentration in mg/mL by 1000 to obtain µg/mL. For example, 2 mg/mL equals 2000 µg/mL.

5. What is the default cuvette pathlength and can I change it?

The default pathlength is 1 cm, which corresponds to most standard cuvettes. You can adjust this value in the calculator if your cuvette has a different internal width.

How to Use

Select a protein or choose custom to enter your own extinction coefficient and molecular weight.
Enter the absorbance at 280 nm, pathlength, and dilution factor.
Click Calculate to determine the protein concentration.