1. What is the A280 Protein Concentration Calculation?

The A280 method estimates protein concentration using ultraviolet absorbance at 280 nm. This method relies on the absorption properties of aromatic amino acids (tryptophan, tyrosine, and phenylalanine) present in proteins.

2. How Does the Calculator Work?

The calculator uses the standard formula:

Where:

• \( A_{280} \) — Absorbance at 280 nm
• 1.45 — Conversion factor (mg/mL per absorbance unit)

Explanation: The conversion factor of 1.45 is a general approximation that works well for many proteins. For more accurate results, specific extinction coefficients should be used when available.

3. Importance of Protein Concentration Measurement

Details: Accurate protein concentration measurement is essential for various biochemical applications including protein purification, enzyme kinetics studies, protein-protein interaction assays, and quantitative proteomics.

4. Using the Calculator

Tips: Enter the A280 absorbance value measured from your spectrophotometer. Ensure the sample is properly blanked and the measurement is taken at 280 nm wavelength.

5. Frequently Asked Questions (FAQ)

Q1: Why use A280 for protein concentration measurement?
A: A280 is a quick, non-destructive method that doesn't require chemical reagents, making it ideal for monitoring protein purification and handling precious samples.

Q2: What are the limitations of the A280 method?
A: The method assumes average aromatic amino acid content. Proteins with unusual amino acid composition may give inaccurate results. Also, nucleic acid contamination can interfere with measurements.

Q3: When should I use a specific extinction coefficient instead?
A: For precise quantitative work, use the protein's specific extinction coefficient calculated from its amino acid sequence, especially when working with recombinant or well-characterized proteins.

Q4: How do I handle samples with nucleic acid contamination?
A: Use correction formulas (such as A280 - 0.75×A260) or purify the protein further to remove nucleic acid contaminants before measurement.

Q5: What is the typical range of A280 values for accurate measurement?
A: For most spectrophotometers, A280 values between 0.1 and 1.0 provide the most accurate results, within the linear range of the instrument.