1. What is the A280 Protein Concentration Calculation?

The A280 method estimates protein concentration by measuring absorbance at 280 nm, where tryptophan and tyrosine residues absorb UV light. This provides a quick, non-destructive way to quantify protein concentration in solution.

2. How Does the Calculator Work?

The calculator uses the Beer-Lambert law adapted for proteins:

Where:

• \( A_{280} \) — Absorbance at 280 nm (no units)
• \( MW \) — Molecular weight (g/mol)
• \( \epsilon_{280} \) — Molar extinction coefficient at 280 nm (L/mol·cm)

Explanation: The equation converts absorbance to concentration using the protein's specific extinction coefficient and molecular weight.

3. Importance of Protein Concentration Measurement

Details: Accurate protein concentration is essential for experimental reproducibility, protein purification, enzymatic assays, and structural studies.

4. Using the Calculator

Tips: Enter A280 reading (typically 0.1-1.0 for best accuracy), molecular weight in g/mol, and extinction coefficient in L/mol·cm. The extinction coefficient can be calculated from amino acid sequence or measured experimentally.

5. Frequently Asked Questions (FAQ)

Q1: Why use 280 nm for protein measurement?
A: Tryptophan and tyrosine residues have strong absorbance at 280 nm, making it a convenient wavelength for most proteins.

Q2: How accurate is the A280 method?
A: Typically ±10-20% accuracy, depending on protein composition. Methods like Bradford or BCA may be more accurate for some applications.

Q3: What if my protein lacks tryptophan?
A: The method will be less sensitive. Consider using 205 nm absorbance instead, which detects peptide bonds.

Q4: How do I determine the extinction coefficient?
A: It can be calculated from amino acid sequence (using tools like ProtParam) or measured experimentally.

Q5: Does buffer composition affect the measurement?
A: Yes, some buffers (like Tris) absorb at 280 nm. Always blank with your buffer solution.