Introduction
When the concentration of a given protein is known, the concentration and molecular weight can be used to determine the molar extinction coefficient.
Beer-Lambert law states that molar absorptivity is constant and that the absorbance is proportional to concentration for a given substance dissolved in a given solute and measured at a given wavelength. Accordingly, molar absorptivities are commonly called molar extinction coefficients
A = ε ∙ l ∙ c where A is the absorbance at a given wave length (λ)
When l = 1 cm, then A = ε ∙ c
- A Absorbance
- l Light path [cm]
- c Concentration[g/L]
- ε Molar extinction coefficient[L/mol∙cm] (L mol-1cm-1)
The molar extinction coefficient for a particular protein can be determined from the measured absorbance and the specific absorptivity constant as. It corresponds to the slope of the line and is dependent on the matrix (buffer), etc.
Note: Absorptivity measurements can be significantly influenced by the composition, temperature, and pH of the aqueous buffer solution applied to solubilize proteins.
The as can be converted to the molar extinction coefficient using the relationship am = as ∙ MW, where MW is the molecular weight of the protein.
Calculation of protein concentration (light path length 1cm)
Once an absorptivity constant value is determined for a particular protein in a well-defined formulation matrix, the concentration of that protein in solution can be determined from its measured absorbance and the specific absorptivity constant using a derivative of the Beer-Lambert law:
c protein = Absorbance of protein in solution (1-cm path length) / as
c protein [g/L] (g L-1)
as [L/g ∙ cm] (L g-1 cm-1)
What we need from you to perform this analysis:
- 1-3 mg purified protein
- The protein amino acid sequence
- 5 ml of the buffer to use for the measurement
- Concentration range to cover
If you do not provide the buffer for the extinction coefficient measurement we will use as a standard PBS-buffer.
