1. What is A280 Protein Concentration Measurement?

The A280 method measures protein concentration by ultraviolet absorbance at 280 nm, where aromatic amino acids (tryptophan and tyrosine) absorb light. This is a quick, non-destructive method for protein quantification that doesn't require reagents.

2. How Does the Calculator Work?

The calculator uses the Beer-Lambert law:

Where:

• \( C \) — Protein concentration (M)
• \( A_{280} \) — Absorbance at 280 nm (AU)
• \( \epsilon \) — Molar extinction coefficient (M⁻¹cm⁻¹)
• \( l \) — Path length (cm)

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

3. Importance of Accurate Protein Quantification

Details: Accurate protein concentration measurement is essential for experimental reproducibility, proper sample loading in assays, and correct interpretation of experimental results.

4. Using the Calculator

Tips: Enter A280 value (typically between 0.1-1.0 for accurate measurement), the protein's extinction coefficient (available in databases or can be calculated from sequence), and path length (usually 1 cm for standard cuvettes).

5. Frequently Asked Questions (FAQ)

Q1: How do I find my protein's extinction coefficient?
A: It can be calculated from protein sequence using tools like ProtParam, or found in literature for well-characterized proteins.

Q2: What is the typical range for A280 measurements?
A: For best accuracy, keep A280 between 0.1-1.0. Samples outside this range should be diluted or concentrated.

Q3: Can I use this for proteins without tryptophan or tyrosine?
A: No, proteins lacking these aromatic amino acids won't absorb significantly at 280 nm. Consider alternative methods like Bradford assay.

Q4: How does buffer composition affect the measurement?
A: Some buffers (like Tris) absorb at 280 nm. Always blank with your sample buffer and avoid high concentrations of absorbing compounds.

Q5: What if my protein has unusual spectral properties?
A: For proteins with chromophores (e.g., heme proteins), consider alternative quantification methods or correct for additional absorbance.