Protein Molar Concentration Calculator Sigma

Sigma's Molar Concentration Formula:

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

Absorbance at 280 nm (A₂₈₀):

dimensionless

Extinction Coefficient (ε):

M^-1cm^-1

Path Length (l):

Protein Molar Concentration (C):

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1. What is Sigma's Molar Concentration Formula?

Sigma's molar concentration formula calculates protein concentration from absorbance at 280 nm (A₂₈₀), using the protein's extinction coefficient (ε) and the path length (l) of the cuvette. This method is based on the absorbance of aromatic amino acids (tryptophan and tyrosine) in the UV range.

2. How Does the Calculator Work?

The calculator uses Sigma's formula:

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

Where:

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

Explanation: The formula relates the measured absorbance to concentration through Beer-Lambert law, where absorbance is proportional to concentration, path length, and the substance's extinction coefficient.

3. Importance of Protein Concentration

Details: Accurate protein concentration determination is essential for experimental reproducibility, protein purification, enzyme kinetics studies, and biochemical assays.

4. Using the Calculator

Tips: Enter A₂₈₀ value from spectrophotometer, extinction coefficient for your protein (often provided by suppliers or calculated from sequence), and cuvette path length (typically 1 cm). All values must be positive numbers.

5. Frequently Asked Questions (FAQ)

Q1: Where do I find the extinction coefficient for my protein?
A: It's often provided by protein suppliers. Alternatively, it can be calculated from amino acid sequence using tools like ProtParam.

Q2: Why use 280 nm for protein concentration?
A: Tryptophan and tyrosine residues absorb strongly at 280 nm, making this wavelength ideal for most proteins.

Q3: What if my protein lacks tryptophan and tyrosine?
A: Alternative methods like Bradford or BCA assay may be needed for such proteins.

Q4: How accurate is this method?
A: Accuracy depends on knowing the correct extinction coefficient. It's typically ±5-10% if ε is accurate.

Q5: Does this work for protein mixtures?
A: No, this method gives total protein concentration based on aromatic amino acids. For mixtures, other methods may be preferable.