Protein Concentration Calculator A280

Protein Concentration Formula:

\[ \text{Conc (mg/mL)} = \frac{A280}{\varepsilon \times \text{path length}} \]

Protein Concentration:

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1. What is the A280 Protein Concentration Calculation?

The A280 method estimates protein concentration by measuring absorbance at 280nm, where aromatic amino acids (tryptophan and tyrosine) absorb light. This provides a quick, non-destructive way to quantify protein concentration in solution.

2. How Does the Calculator Work?

The calculator uses the Beer-Lambert law:

\[ \text{Conc (mg/mL)} = \frac{A280}{\varepsilon \times \text{path length}} \]

Where:

\( A280 \) — Absorbance at 280nm
\( \varepsilon \) — Extinction coefficient (M⁻¹cm⁻¹)
\( \text{path length} \) — Cuvette path length (cm)

Explanation: The equation relates the absorption of light to the properties of the material through which the light is traveling.

3. Importance of Protein Concentration Measurement

Details: Accurate protein quantification is essential for various biochemical applications including enzyme kinetics, protein purification, Western blotting, and ensuring consistent results in experiments.

4. Using the Calculator

Tips: Enter absorbance at 280nm, the protein's extinction coefficient, and the path length of your cuvette. Standard cuvettes typically have a 1cm path length.

5. Frequently Asked Questions (FAQ)

Q1: What is a typical extinction coefficient for proteins?
A: Extinction coefficients vary by protein but typically range from 10,000 to 50,000 M⁻¹cm⁻¹. You can calculate it from the protein's amino acid sequence.

Q2: Why use 280nm for protein measurement?
A: Tryptophan and tyrosine residues have strong absorbance at 280nm, making it a convenient wavelength for protein quantification.

Q3: What are the limitations of the A280 method?
A: The method assumes the protein contains tryptophan or tyrosine. Nucleic acid contamination can interfere as they also absorb at 260-280nm.

Q4: How do I determine my protein's extinction coefficient?
A: You can calculate it theoretically from the amino acid sequence using online tools like ProtParam, or measure it experimentally.

Q5: What if my sample is too concentrated?
A: Dilute your sample until the absorbance falls within the linear range of your spectrophotometer (typically 0.1-1.0 AU).