1. What is the Protein Concentration Equation?

The protein concentration equation estimates protein concentration from absorbance at 280 nm (A₂₈₀), using the protein's extinction coefficient and the path length of the measurement. This method is based on the absorbance of aromatic amino acids (tryptophan and tyrosine) in proteins.

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 (unitless)
• \( \epsilon \) — Extinction coefficient (mL/(mg·cm))
• \( l \) — Path length of the cuvette (cm)

Explanation: The equation relates the measured absorbance to protein concentration through the Beer-Lambert law, where absorbance is proportional to concentration, path length, and the extinction coefficient.

3. Importance of Protein Concentration Measurement

Details: Accurate protein concentration measurement is essential for protein purification, enzymatic assays, protein-protein interaction studies, and many other biochemical experiments. The A₂₈₀ method is quick and non-destructive, allowing for subsequent use of the sample.

4. Using the Calculator

Tips:

• Measure A₂₈₀ with a spectrophotometer using a quartz cuvette
• Use the correct extinction coefficient for your protein (often provided in literature)
• Standard path length is 1 cm (adjust if using different cuvette)
• Ensure the sample is free of light-scattering particles which can affect absorbance

5. Frequently Asked Questions (FAQ)

Q1: How do I find the extinction coefficient for my protein?
A: The extinction coefficient can be calculated from the protein sequence (using tools like ProtParam) or measured experimentally. It's often provided with purified proteins.

Q2: What if my protein doesn't have tryptophan or tyrosine?
A: The A₂₈₀ method won't work well. Consider alternative methods like Bradford, BCA, or Lowry assays.

Q3: Why is the path length important?
A: Absorbance is directly proportional to path length. Most extinction coefficients are given for 1 cm path length - adjust if using a different cuvette.

Q4: What are typical extinction coefficient values?
A: They vary widely (e.g., 0.1-3.0 mL/(mg·cm)). For antibodies, ~1.4 mL/(mg·cm) is common.

Q5: What are limitations of this method?
A: Nucleic acid contamination can interfere (they also absorb at 280 nm). The method is less accurate for very dilute or concentrated samples.