1. What is the A280 Protein Concentration Calculation?

The A280 method estimates protein concentration using UV absorbance at 280 nm, which detects aromatic amino acids (tryptophan, tyrosine, and phenylalanine). This method provides a quick and non-destructive way to measure protein concentration in solution.

2. How Does the Calculator Work?

The calculator uses the Beer-Lambert law based formula:

Where:

• \( A280 \) — Absorbance at 280 nm
• \( \varepsilon \) — Molar extinction coefficient (M⁻¹cm⁻¹)
• 0.1 — Pathlength correction factor for standard 1 cm cuvette

Explanation: The equation calculates protein concentration based on the specific absorbance characteristics of the protein's aromatic amino acids at 280 nm wavelength.

3. Importance of Protein Concentration Measurement

Details: Accurate protein concentration measurement is essential for biochemical experiments, protein purification, enzyme kinetics studies, and ensuring consistent results in research and diagnostic applications.

4. Using the Calculator

Tips: Enter absorbance reading at 280 nm and the protein's molar extinction coefficient. Ensure measurements are taken with properly calibrated spectrophotometers using standard 1 cm pathlength cuvettes.

5. Frequently Asked Questions (FAQ)

Q1: What is the molar extinction coefficient?
A: The molar extinction coefficient (ε) is a measure of how strongly a chemical species absorbs light at a given wavelength. For proteins, it's typically in the range of 10,000-50,000 M⁻¹cm⁻¹.

Q2: How do I determine the extinction coefficient for my protein?
A: Extinction coefficients can be calculated from the protein's amino acid sequence using online tools or measured experimentally using known concentrations of purified protein.

Q3: What are the limitations of the A280 method?
A: This method requires proteins with aromatic amino acids. It can be affected by nucleic acid contamination and doesn't work well for proteins with few aromatic residues.

Q4: Can I use this for any pathlength?
A: The formula assumes a 1 cm pathlength. For other pathlengths, adjust the calculation accordingly (A280/pathlength in cm).

Q5: What is the typical concentration range for this method?
A: The A280 method is generally reliable for protein concentrations between 0.1-1.0 mg/mL, though this varies depending on the specific protein's extinction coefficient.