1. What is the A280 Protein Concentration Calculation?

The A280 method estimates protein concentration based on the absorbance of ultraviolet light at 280nm wavelength. This technique relies on the presence of aromatic amino acids (tryptophan and tyrosine) in proteins that absorb light at this specific wavelength.

2. How Does the Calculator Work?

The calculator uses the standard formula:

Where:

• \( A280 \) — Absorbance reading at 280nm
• \( 1.55 \) — Extinction coefficient for average proteins (mg⁻¹·mL·cm⁻¹)

Explanation: This calculation assumes an average protein composition with typical amounts of tryptophan and tyrosine. The extinction coefficient of 1.55 is derived from the average absorbance properties of most proteins.

3. Importance of Protein Concentration Measurement

Details: Accurate protein concentration measurement is essential for various biochemical applications including enzyme kinetics studies, protein purification, Western blotting, and ensuring consistent results in experimental protocols.

4. Using the Calculator

Tips: Enter the absorbance reading at 280nm (A280) obtained from a spectrophotometer. Ensure the sample is properly diluted to fall within the linear range of the instrument (typically 0.1-1.0 AU).

5. Frequently Asked Questions (FAQ)

Q1: Why use the A280 method for protein quantification?
A: The A280 method is quick, non-destructive, and doesn't require additional reagents, making it ideal for routine protein concentration measurements.

Q2: Are there limitations to this method?
A: Yes, this method is less accurate for proteins with unusual amino acid compositions, particularly those with very high or very low amounts of aromatic amino acids.

Q3: What factors can interfere with A280 measurements?
A: Nucleic acid contamination, turbidity from particulate matter, and certain buffer components can interfere with accurate A280 measurements.

Q4: When should I use a different quantification method?
A: Consider Bradford, BCA, or Lowry assays when working with proteins that have atypical aromatic amino acid content or when contaminants interfere with UV absorbance.

Q5: How should I prepare samples for A280 measurement?
A: Use an appropriate buffer blank for reference, ensure samples are clear without turbidity, and dilute samples to fall within the instrument's linear range.