1. What is A280 Protein Concentration Measurement?

The A280 method estimates protein concentration by measuring absorbance at 280 nm, where tryptophan and tyrosine residues absorb UV light. This direct spectrophotometric method is simple, rapid, and non-destructive, making it ideal for routine protein quantification.

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)
• \( \epsilon \) — Extinction coefficient (mL·mg^-1·cm^-1)
• \( l \) — Path length (cm)

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

3. Importance of Accurate Protein Quantification

Details: Precise protein concentration measurement is essential for experimental reproducibility, protein purification, enzyme assays, and preparing samples for downstream applications like SDS-PAGE or mass spectrometry.

4. Using the Calculator

Tips:

• Measure A280 against an appropriate blank (typically buffer alone)
• Use the protein's theoretical or experimentally determined extinction coefficient
• Standard cuvettes typically have 1 cm path length (use actual measured value if different)
• For best results, A280 should be between 0.1 and 1.0 AU

5. Frequently Asked Questions (FAQ)

Q1: How do I find the extinction coefficient for my protein?
A: Use tools like ProtParam (ExPASy) with the protein sequence, or measure it experimentally using known concentrations.

Q2: What if my protein lacks tryptophan/tyrosine?
A: A280 won't work well - consider alternative methods like Bradford, BCA, or Lowry assays.

Q3: Why is my calculated concentration inaccurate?
A: Common issues include: incorrect ε value, nucleic acid contamination (absorbs at 280 nm), or light-scattering particles.

Q4: How does buffer composition affect A280?
A: Some buffers (e.g., Tris, DTT) absorb at 280 nm - always blank with the same buffer.

Q5: Can I use this for protein mixtures?
A: Yes, but the result will be an average concentration based on the mixture's average ε.