Protein Concentration Calculator

Using the Protein Concentration Calculator allows you to determine the exact quantity of protein in a sample without the need for destructive dyeing or time-consuming standard curves. Follow these laboratory steps:

Step 1: Set your spectrophotometer to the 280 nm wavelength.

Step 2: Measure the Absorbance (A₂₈₀) of your protein sample. Ensure you have "blanked" the machine using the same buffer the protein is dissolved in.

Step 3: Enter the A₂₈₀ value into the first field.

Step 4: Enter the Extinction Coefficient (ε) for your specific protein. If you are measuring a generic mixture, 1.0 is a common estimate, but for pure proteins like BSA or IgG, you should use the specific validated constant.

Step 5: Enter the Path Length of your cuvette (standard is 1.0 cm).

Step 6: Enter the Dilution Factor if you diluted the sample to keep the absorbance within the linear range of the machine (A < 1.5).

Step 7: Click "Calculate."

Step 8: Review the Concentration in mg/mL. This value is critical for ensuring you load the correct amount of protein onto an electrophoresis gel or into a downstream biochemical assay.

The Protein Concentration Calculator is based on the Beer-Lambert Law, which describes how light is absorbed by molecules in a solution.

The fundamental formula is: Concentration (c) = A / (ε × l)

Where: - A = Measured Absorbance at 280 nm. - ε = Molar Extinction Coefficient (a constant representing how much light a specific protein absorbs). - l = Path length of the light through the sample (typically 1 cm).

Unlike DNA, which has a very consistent absorbance, different proteins have different extinction coefficients because A₂₈₀ absorbance is primarily driven by aromatic amino acids—specifically Tryptophan and Tyrosine. A protein with many Tryptophan residues will absorb light much more strongly than one with none.

To account for dilution, the final formula used by the calculator is: Final Concentration = (A / (ε × l)) × Dilution Factor

Example: A sample of IgG (ε = 1.35) gives an absorbance of 0.54. 0.54 / (1.35 × 1) = 0.4 mg/mL. If the sample was diluted 1:10 before measuring, the original concentration was 4.0 mg/mL. This method is exceptionally fast and non-destructive, meaning you can use the sample for other experiments after measurement, provided the UV exposure was brief.

Protein concentration is a fundamental value in biochemistry and molecular biology. Knowing how much protein you have in a sample is essential before running assays, preparing gels, or adding reagents in any reaction — using too little gives weak results, and too much can saturate or interfere with measurements.

This calculator uses spectrophotometric quantification at 280 nm, which exploits the fact that aromatic amino acids (primarily tryptophan and tyrosine) absorb UV light at this wavelength. The technique is fast, requires no additional reagents, and is non-destructive to the sample. However, it depends on five key input values:

Absorbance (A280): Measured with a spectrophotometer. Captures how much UV light the sample absorbs at 280 nm, where most proteins show their peak absorption.

Extinction Coefficient (ε): A protein-specific constant that quantifies how strongly the molecule absorbs light. It varies based on the amino acid composition and must be looked up or calculated for each protein.

Path Length: The inner diameter of the cuvette used — almost always 1 cm in standard lab setups.

Molecular Mass: The atomic mass of the protein in g/mol, needed to convert from molar units to mg/mL.

Dilution Factor: Applied when the original sample was diluted before measurement. Multiply absorbance by this number to recover the original concentration.

Key Reference Values: Some commonly used extinction coefficients and molecular masses — BSA: ε = 43,824 M⁻¹cm⁻¹, MW = 66,463 g/mol. IgG: ε = 210,000 M⁻¹cm⁻¹, MW = 150,000 g/mol. Lysozyme: ε = 37,901 M⁻¹cm⁻¹, MW = 14,000 g/mol. Streptavidin: ε = 176,000 M⁻¹cm⁻¹, MW = 55,000 g/mol. These values are validated for measurements at 280 nm.