1. What is the A280 Protein Concentration Method?

The A280 method estimates protein concentration by measuring absorbance at 280 nm, where tryptophan and tyrosine residues absorb UV light. This direct method is simple and doesn't require additional reagents.

2. How Does the Calculator Work?

The calculator uses the Beer-Lambert law:

Where:

• \( A_{280} \) — Absorbance at 280 nm (dimensionless)
• \( \varepsilon_{280} \) — Extinction coefficient (M^-1cm^-1)
• \( path\_length \) — Cuvette path length (cm)

Explanation: The equation relates absorbance to concentration through the extinction coefficient and path length.

3. Importance of Protein Quantification

Details: Accurate protein concentration measurement is essential for experimental reproducibility, protein purification, enzyme kinetics, and many biochemical assays.

4. Using the Calculator

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

5. Frequently Asked Questions (FAQ)

Q1: What are typical extinction coefficients?
A: Most proteins have ε₂₈₀ between 10,000-50,000 M^-1cm^-1. Antibodies are often ~210,000 M^-1cm^-1.

Q2: What are the limitations of A280 measurement?
A: Nucleic acid contamination can interfere. The method requires known extinction coefficient and is less accurate for very dilute or concentrated samples.

Q3: How do I determine my protein's extinction coefficient?
A: Use tools like ProtParam (ExPASy) with the protein sequence, or measure it experimentally using known concentrations.

Q4: What's the ideal A280 range for measurement?
A: 0.1-1.0 absorbance units for most spectrophotometers. For values outside this range, dilute or use a shorter path length cuvette.

Q5: Can I use this for protein mixtures?
A: Only if you know the average extinction coefficient. For complex mixtures, other methods like Bradford assay may be better.