1. What is A280 Protein Concentration?

The A280 method estimates protein concentration by measuring absorbance at 280 nm, where tryptophan and tyrosine residues absorb UV light. This quick, non-destructive method is widely used in biochemistry and molecular biology.

2. How Does the Calculator Work?

The calculator uses the Beer-Lambert law:

Where:

• \( C \) — Protein concentration (mg/mL)
• \( A_{280} \) — Absorbance at 280 nm (AU)
• \( \varepsilon \) — Extinction coefficient (mL/mg/cm)
• \( l \) — Path length (cm)

Explanation: The absorbance is directly proportional to the protein concentration, extinction coefficient, and path length of the cuvette.

3. Importance of Protein Quantification

Details: Accurate protein concentration measurement is essential for experimental reproducibility, protein purification, enzyme assays, and Western blot normalization.

4. Using the Calculator

Tips: Enter the measured A280 value, the protein's extinction coefficient (often provided in protein databases or literature), and the cuvette path length (typically 1 cm). All values must be positive numbers.

5. Frequently Asked Questions (FAQ)

Q1: Where do I find the extinction coefficient?
A: It can be calculated from amino acid sequence (using tools like ProtParam) or found in literature for well-characterized proteins.

Q2: What if my protein has unusual absorbance?
A: Proteins lacking tryptophan/tyrosine may need alternative methods like Bradford or BCA assays.

Q3: What's a typical extinction coefficient?
A: Most proteins have ε values between 0.5-2.0 mL/mg/cm. Antibodies often have ε ~1.4 mL/mg/cm.

Q4: How accurate is this method?
A: ±10-20% accuracy, depending on protein composition and buffer conditions. More accurate for purified proteins than complex mixtures.

Q5: What if I used a different path length?
A: Adjust the path length value accordingly (e.g., 0.5 cm for half-size cuvettes).