1. What is the PPP Method?

The PPP (Protein Purification Protocol) method estimates protein concentration using UV absorbance at 280nm, which primarily detects aromatic amino acids (tryptophan and tyrosine). This is a quick and non-destructive method for 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 280nm (dimensionless)
• \( \epsilon \) — Extinction coefficient (ml mg^-1 cm^-1)
• \( l \) — Path length (cm)

Explanation: The absorbance is directly proportional to the protein concentration when measured at the appropriate wavelength.

3. Importance of Protein Concentration

Details: Accurate protein concentration measurement is essential for experimental reproducibility, protein purification monitoring, and proper sample preparation for downstream applications.

4. Using the Calculator

Tips: Enter A280 value (typically between 0.1-1.0 for accurate measurement), the protein-specific extinction coefficient, and the cuvette path length (usually 1 cm).

5. Frequently Asked Questions (FAQ)

Q1: How do I determine the extinction coefficient?
A: It can be calculated from the protein sequence or measured experimentally. Online tools like ProtParam can calculate it from amino acid composition.

Q2: What if my protein has few aromatic amino acids?
A: The PPP method may underestimate concentration. Consider alternative methods like Bradford or BCA assays.

Q3: What's a typical extinction coefficient range?
A: Most proteins range from 0.5-2.0 ml mg^-1 cm^-1, but it varies widely based on aromatic amino acid content.

Q4: Why is 280nm used?
A: Tryptophan and tyrosine absorb strongly at this wavelength, while nucleic acid contamination absorbs more at 260nm.

Q5: How accurate is this method?
A: Typically ±10-20% accuracy, assuming proper blanking and no interfering substances.