1. What is the A280 Protein Concentration Equation?

The A280 method estimates protein concentration by measuring absorbance at 280 nm, which is primarily due to tryptophan and tyrosine residues. The fat correction (FC) accounts for light scattering by lipid particles that can interfere with absorbance measurements.

2. How Does the Calculator Work?

The calculator uses the protein concentration equation:

Where:

• \( C \) — Protein concentration (mg/ml)
• \( A_{280} \) — Absorbance at 280 nm (dimensionless)
• \( \epsilon \) — Extinction coefficient (ml mg^-1 cm^-1)
• \( l \) — Path length (cm)
• \( FC \) — Fat correction (mg/ml)

Explanation: The equation first calculates the apparent protein concentration from absorbance, then subtracts the contribution from light scattering by fat particles.

3. Importance of Fat Correction

Details: In samples containing lipids or fat, light scattering can artificially increase A280 readings. The fat correction accounts for this interference to provide more accurate protein concentration measurements.

4. Using the Calculator

Tips:

• Measure A280 with a spectrophotometer using appropriate blank
• Use the extinction coefficient specific to your protein
• Standard path length is 1 cm (adjust if using different cuvette)
• Determine fat correction empirically from lipid-only control

5. Frequently Asked Questions (FAQ)

Q1: How do I determine the extinction coefficient?
A: Calculate from protein sequence (theoretical) or measure experimentally using known concentrations.

Q2: When is fat correction necessary?
A: For samples containing >0.5% lipids, membrane preparations, or any visibly turbid solutions.

Q3: What's a typical extinction coefficient?
A: For antibodies ~1.4 ml mg^-1 cm^-1; varies greatly by protein (0.5-2.5 range common).

Q4: How accurate is this method?
A: ±10-20% typically, assuming proper blanking and known extinction coefficient.

Q5: Are there alternatives to A280 measurement?
A: Yes, Bradford, BCA, or Lowry assays may be better for lipid-rich samples.