Calculating Protein Concentration From Absorbance 280 Nm

Protein Concentration Formula:

\[ \text{Conc (mg/mL)} = \frac{A_{280}}{\varepsilon \times \text{path length} \times 1.55} \]

Protein Concentration:

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1. What Is Protein Concentration Calculation From A280?

The calculation of protein concentration from absorbance at 280 nm is a widely used method in biochemistry and molecular biology. It leverages the property that aromatic amino acids (tryptophan, tyrosine, and phenylalanine) in proteins absorb ultraviolet light at 280 nm.

2. How Does The Calculator Work?

The calculator uses the formula:

\[ \text{Conc (mg/mL)} = \frac{A_{280}}{\varepsilon \times \text{path length} \times 1.55} \]

Where:

\( A_{280} \) — Absorbance at 280 nm
\( \varepsilon \) — Extinction coefficient (M⁻¹cm⁻¹)
path length — Cuvette path length in centimeters
1.55 — Conversion factor for mg/mL concentration

Explanation: This equation converts the measured absorbance into protein concentration by accounting for the protein's specific extinction coefficient and the measurement conditions.

3. Importance Of Protein Concentration Measurement

Details: Accurate protein concentration measurement is essential for various applications including protein purification, enzyme kinetics studies, western blotting, and preparing samples for structural biology techniques.

4. Using The Calculator

Tips: Enter the absorbance reading at 280 nm, the protein's extinction coefficient, and the path length of your cuvette. All values must be positive numbers.

5. Frequently Asked Questions (FAQ)

Q1: Why use 280 nm for protein concentration measurement?
A: 280 nm is used because aromatic amino acids in proteins have strong absorbance at this wavelength, making it a reliable indicator of protein concentration.

Q2: How do I determine the extinction coefficient for my protein?
A: The extinction coefficient can be calculated from the protein's amino acid sequence using online tools or measured experimentally.

Q3: What is the significance of the path length?
A: The path length is the distance light travels through the sample. Standard cuvettes typically have a 1 cm path length, but this should be verified for accurate calculations.

Q4: Are there limitations to this method?
A: This method assumes the protein contains typical amounts of aromatic amino acids. Proteins with unusual amino acid composition may give inaccurate results.

Q5: Can this method be used for all protein types?
A: While widely applicable, some proteins with few aromatic residues or those that aggregate may require alternative concentration determination methods.