Calculate Protein Concentration From A280

Protein Concentration Equation:

\[ C = \frac{A280}{\epsilon} \times \text{dilution factor} \]

Absorbance at 280 nm (A280):

absorbance units

Extinction coefficient (ε):

mL/(mg·cm)

Dilution factor:

fold

Path length:

Protein Concentration (C):

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1. What is the A280 Protein Concentration Method?

The A280 method estimates protein concentration by measuring absorbance at 280 nm, where aromatic amino acids (tryptophan and tyrosine) absorb light. This direct UV method is simple, non-destructive, and doesn't require additional reagents.

2. How Does the Calculator Work?

The calculator uses the Beer-Lambert law adapted for proteins:

\[ C = \frac{A280}{\epsilon \times l} \times \text{dilution factor} \]

Where:

\( C \) — Protein concentration (mg/mL)
\( A280 \) — Absorbance at 280 nm (unitless)
\( \epsilon \) — Extinction coefficient (mL/(mg·cm))
\( l \) — Path length (cm)
dilution factor — Sample dilution (fold)

Explanation: The equation relates absorbance to concentration using the protein's specific extinction coefficient, which depends on its amino acid composition.

3. Importance of Accurate Protein Quantification

Details: Precise protein concentration measurement is essential for experimental reproducibility, proper sample loading in assays like SDS-PAGE or Western blotting, and accurate biochemical studies.

4. Using the Calculator

Tips:

Measure A280 in a spectrophotometer with quartz cuvettes
Use the protein's theoretical or experimentally determined extinction coefficient
Account for any sample dilution
Standard path length is 1 cm (adjust if using different cuvettes)

5. Frequently Asked Questions (FAQ)

Q1: How do I find my protein's extinction coefficient?
A: Use tools like ProtParam (ExPASy) with your protein sequence, or measure it experimentally using known concentrations.

Q2: What are typical extinction coefficients?
A: Most proteins have ε between 0.5-2.0 mL/(mg·cm). Antibodies are typically ~1.4 mL/(mg·cm).

Q3: When is this method not appropriate?
A: For samples with high nucleic acid contamination or when the protein lacks tryptophan/tyrosine.

Q4: What's a good A280 range to measure?
A: Ideally 0.1-1.0 absorbance units. Higher values may require dilution for accurate measurement.

Q5: How does path length affect results?
A: The standard equation assumes 1 cm path length. For microvolume measurements with shorter path lengths, you must enter the actual path length.