1. What is the A280 Protein Concentration Method?

The A280 method estimates protein concentration by measuring absorbance at 280nm, where tryptophan and tyrosine residues absorb UV light. This direct spectrophotometric method is simple and non-destructive, requiring only the protein sample and a UV spectrophotometer.

2. How Does the Calculator Work?

The calculator uses the Beer-Lambert law equation:

Where:

• \( C \) — Protein concentration (mg/mL)
• \( A_{280} \) — Absorbance at 280nm (unitless)
• \( \epsilon \) — 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 downstream applications like SDS-PAGE or Western blotting.

4. Using the Calculator

Tips:

• Measure A280 in a quartz cuvette with appropriate buffer blank
• Use the extinction coefficient specific to your protein
• Standard path length is 1cm (adjust if using different cuvette)
• Ideal A280 range: 0.1-1.0 (dilute if above 1.0)

5. Frequently Asked Questions (FAQ)

Q1: How do I find my protein's extinction coefficient?
A: Calculate from amino acid sequence (ProtParam tool) or measure experimentally using dry weight.

Q2: Why is my A280 measurement inaccurate?
A: Common issues include nucleic acid contamination (use A260/A280 ratio), buffer absorbance, or light-scattering from aggregates.

Q3: What if my protein lacks tryptophan/tyrosine?
A: Use alternative methods like Bradford, BCA, or Lowry assays which don't rely on aromatic residues.

Q4: How does path length affect the measurement?
A: Shorter path lengths allow measurement of more concentrated samples without dilution.

Q5: What's a typical extinction coefficient?
A: Most proteins have ε ~0.5-1.5 mL/mg/cm. Antibodies often have ε ~1.4 mL/mg/cm.