Calculated Concentrations
| Unit Type | Value | Unit |
|---|
Absorbance Visualizer
A280 Protein Concentration
Calculate Protein Concentration with A280
The Protein Concentration Calculator provides a quick and accurate method to determine the concentration of purified protein samples using absorbance measurements at 280 nm. This method relies on the intrinsic absorbance of aromatic amino acids (tryptophan, tyrosine, and phenylalanine) and is widely used in biochemistry and molecular biology laboratories for protein quantification.
The Beer-Lambert Law
This calculator utilizes the Beer-Lambert law, which establishes a linear relationship between absorbance and concentration of an absorbing species. The formula used for calculation is:
\(A = \varepsilon \cdot l \cdot c\)
Where:
- A is the Absorbance measured at 280 nm (dimensionless).
- ε (Epsilon) is the Extinction Coefficient (molar attenuation coefficient), typically in units of \(M^{-1}cm^{-1}\) or \((mg/mL)^{-1}cm^{-1}\).
- l is the Path Length of the light through the sample, usually equal to the width of the cuvette (standard is 1 cm).
- c is the Concentration of the protein sample.
To solve for concentration, the formula is rearranged:
\(c = \frac{A}{\varepsilon \cdot l}\)
Extinction Coefficients and Units
The accuracy of this calculation depends heavily on the correct value of the extinction coefficient.
- Molar Extinction Coefficient (\(M^{-1}cm^{-1}\)): Used when calculating molarity (M). It can be estimated based on the protein sequence using the number of Tryptophan, Tyrosine, and Cystine residues.
- Mass Extinction Coefficient (\((mg/mL)^{-1}cm^{-1}\)): Also referred to as the Absorbance of a 0.1% solution (A280 1mg/mL). This allows for direct calculation of concentration in mg/mL.
If you have the molar extinction coefficient but need the result in mg/mL, the calculator uses the Molecular Weight (MW) to perform the conversion:
\(Concentration_{mg/mL} = Concentration_{Molar} \times MW_{g/mol}\)
Important Considerations
While A280 is a non-destructive and rapid method, several factors can influence accuracy:
- Nucleic Acid Contamination: DNA and RNA absorb significantly at 260 nm but also have absorbance at 280 nm. A high A260/A280 ratio may indicate contamination, skewing the protein concentration results.
- Buffer Composition: Some buffers or additives (like detergents) may absorb UV light. Always blank the spectrophotometer with the exact buffer used for the sample.
- Scattering: Particulates in the solution can cause light scattering, artificially increasing the absorbance reading. Centrifugation or filtration prior to measurement is recommended.