1. What is the Beer-Lambert Law?

The Beer-Lambert law relates the absorption of light to the properties of the material through which the light is traveling. For proteins, it's commonly used to determine concentration from absorbance at 280 nm (A₂₈₀).

2. How Does the Calculator Work?

The calculator uses the Beer-Lambert law:

Where:

• \( C \) — Protein concentration (M)
• \( A_{280} \) — Absorbance at 280 nm (AU)
• \( \epsilon \) — Extinction coefficient (M^-1cm^-1)
• \( l \) — Path length (cm)

Explanation: The equation shows that concentration is directly proportional to absorbance and inversely proportional to the extinction coefficient and path length.

3. Importance of Protein Concentration Measurement

Details: Accurate protein concentration measurement is essential for experimental reproducibility, protein purification, enzyme kinetics studies, and many other biochemical applications.

4. Using the Calculator

Tips:

• Measure A₂₈₀ with a spectrophotometer using quartz cuvettes
• Use the correct extinction coefficient for your protein (often provided in literature)
• Standard path length is 1 cm, but measure actual path length if using different cuvettes
• For best results, dilute samples to keep A₂₈₀ between 0.1 and 1.0

5. Frequently Asked Questions (FAQ)

Q1: Why measure at 280 nm?
A: Proteins absorb UV light at 280 nm due to tryptophan and tyrosine residues in their structure.

Q2: How do I find the extinction coefficient?
A: It can be calculated from the protein sequence (using tools like ProtParam) or measured experimentally.

Q3: What if my protein doesn't have tryptophan or tyrosine?
A: Alternative methods like Bradford or BCA assay should be used in such cases.

Q4: How accurate is this method?
A: It's generally accurate to ±10% when using the correct extinction coefficient and proper sample preparation.

Q5: What are common sources of error?
A: Turbidity, nucleic acid contamination, incorrect path length, and protein aggregation can all affect measurements.