Protein Concentration Calculation

Protein Concentration Formula:

\[ C = \frac{A_{280}}{\epsilon \times l} \]

Absorbance at 280nm (A₂₈₀):

absorbance units

Extinction Coefficient (ε):

ml mg^-1 cm^-1

Path Length (l):

Protein Concentration (C):

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1. What is Protein Concentration Calculation?

The protein concentration calculation using absorbance at 280nm is a common method for estimating protein concentration in solution. It's based on the Beer-Lambert law and the absorbance properties of aromatic amino acids (tryptophan, tyrosine) and disulfide bonds.

2. How Does the Calculator Work?

The calculator uses the following equation:

\[ C = \frac{A_{280}}{\epsilon \times l} \]

Where:

\( C \) — Protein concentration (mg/ml)
\( A_{280} \) — Absorbance at 280nm (dimensionless)
\( \epsilon \) — Extinction coefficient (ml mg^-1 cm^-1)
\( l \) — Path length (cm)

Explanation: The equation relates the absorbance of light by protein solution to its concentration, accounting for the specific protein's light absorption characteristics and the path length of the measurement.

3. Importance of Protein Concentration

Details: Accurate protein concentration measurement is essential for experimental reproducibility, protein purification, enzyme assays, and many biochemical applications.

4. Using the Calculator

Tips: Enter absorbance at 280nm (typically between 0.1-1.0 for accurate measurements), the protein's extinction coefficient (specific to each protein), and the path length of your cuvette (usually 1 cm).

5. Frequently Asked Questions (FAQ)

Q1: Where do I find the extinction coefficient for my protein?
A: The extinction coefficient can be calculated from the protein sequence (using tools like ProtParam) or found in literature for well-characterized proteins.

Q2: Why use 280nm for protein measurement?
A: Aromatic amino acids (tryptophan, tyrosine) and disulfide bonds absorb strongly at 280nm, making it a convenient wavelength for protein detection.

Q3: What are typical extinction coefficients?
A: Extinction coefficients vary widely (e.g., 0.5-2.0 ml mg^-1 cm^-1). For example, bovine serum albumin has ε = 0.66 ml mg^-1 cm^-1.

Q4: What if my protein has few aromatic amino acids?
A: The method may underestimate concentration. Alternative methods like Bradford or BCA assay may be more appropriate.

Q5: How does path length affect the measurement?
A: Standard cuvettes have 1 cm path length. If using different path lengths (e.g., microvolume spectrophotometers), adjust accordingly.