Calculating Protein Concentration From Absorbance And Extinction Coefficient

Beer-Lambert Law for Proteins:

\[ \text{Conc (mg/mL)} = \frac{\text{Absorbance}}{\varepsilon \times \text{path length}} \]

Protein Concentration:

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1. What is the Beer-Lambert Law?

The Beer-Lambert law describes the relationship between the concentration of a substance and the amount of light it absorbs. For proteins, it provides a direct method to calculate concentration from absorbance measurements using the extinction coefficient.

2. How Does the Calculator Work?

The calculator uses the Beer-Lambert equation:

\[ \text{Conc (mg/mL)} = \frac{\text{Absorbance}}{\varepsilon \times \text{path length}} \]

Where:

Conc — Protein concentration (mg/mL)
Absorbance — Measured absorbance value (unitless)
ε — Extinction coefficient (mL/mg/cm)
path length — Cuvette path length (cm)

Explanation: The equation calculates protein concentration based on the light absorption properties of the protein sample at a specific wavelength.

3. Importance of Protein Concentration Measurement

Details: Accurate protein concentration measurement is essential for various biochemical applications including protein purification, enzyme kinetics studies, Western blotting, and protein quantification assays.

4. Using the Calculator

Tips: Enter absorbance value (typically measured at 280nm), extinction coefficient (specific to your protein), and path length (usually 1.0 cm for standard cuvettes). All values must be positive numbers.

5. Frequently Asked Questions (FAQ)

Q1: What is the typical extinction coefficient for proteins?
A: Extinction coefficients vary by protein. Common values range from 0.5 to 2.0 mL/mg/cm. The coefficient is often calculated from the protein's amino acid composition.

Q2: Why measure at 280nm?
A: Proteins absorb light at 280nm due to tryptophan and tyrosine residues, making this wavelength ideal for protein concentration determination.

Q3: What if my absorbance reading is too high?
A: Absorbance values should typically be between 0.1 and 1.0 for accurate measurements. If higher, dilute your sample and multiply the result by the dilution factor.

Q4: How do I find the extinction coefficient for my protein?
A: Extinction coefficients can be calculated from the protein sequence using online tools, found in literature, or measured experimentally.

Q5: Does this work for all proteins?
A: The method works best for proteins containing tryptophan and tyrosine. Proteins lacking these residues may require alternative quantification methods.