1. What is Protein Concentration Calculation?

The protein concentration calculation using absorbance at 280 nm (A₂₈₀) is a common method for determining protein concentration in solution. It relies on the absorbance properties of aromatic amino acids (tryptophan, tyrosine, and phenylalanine) in proteins.

2. How Does the Calculator Work?

The calculator uses the Beer-Lambert law equation:

Where:

• \( A_{280} \) — Absorbance at 280 nm
• \( \epsilon \) — Extinction coefficient (M^-1cm^-1)
• \( pathlength \) — Path length of the cuvette (cm)

Explanation: The equation relates the absorbance of light by a solution to the concentration of the absorbing species in that solution.

3. Importance of Protein Concentration

Details: Accurate protein concentration measurement is essential for experiments involving protein purification, enzyme kinetics, protein-protein interactions, and many other biochemical assays.

4. Using the Calculator

Tips: Enter the absorbance reading at 280 nm, the extinction coefficient for your protein (often provided in literature), and the path length of your cuvette (typically 1 cm).

5. Frequently Asked Questions (FAQ)

Q1: What if my protein doesn't have tryptophan or tyrosine?
A: The A₂₈₀ method won't work well. Consider alternative methods like Bradford or BCA assays.

Q2: How do I find my protein's extinction coefficient?
A: It can be calculated from the protein sequence using tools like ProtParam or found in literature for known proteins.

Q3: What are typical extinction coefficient values?
A: They vary widely but often range from 10,000 to 50,000 M^-1cm^-1 for most proteins.

Q4: Why is path length important?
A: Absorbance is directly proportional to path length. Standard cuvettes are 1 cm, but microvolume instruments may use shorter paths.

Q5: What if my sample is turbid or has contaminants?
A: Turbidity or contaminating nucleic acids can interfere. Consider sample clarification or correction methods.