Protein Concentration Calculator 280 Abs

Beer-Lambert Law:

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

Protein Concentration (C):

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1. What is the 280nm Absorbance Method?

The 280nm absorbance method estimates protein concentration by measuring the absorbance of aromatic amino acids (tryptophan, tyrosine, and phenylalanine) at 280nm. This is a quick, non-destructive method that doesn't require any reagents or protein modification.

2. How Does the Calculator Work?

The calculator uses the Beer-Lambert law:

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

Where:

\( C \) — Protein concentration (mg/mL)
\( A_{280} \) — Absorbance at 280nm (unitless)
\( \epsilon \) — Extinction coefficient (mL/mg/cm)
\( l \) — Path length (cm)

Explanation: The absorbance is directly proportional to the protein concentration, extinction coefficient, and path length of the cuvette.

3. Importance of Protein Quantification

Details: Accurate protein concentration measurement is essential for experimental reproducibility, sample preparation, enzymatic assays, and protein characterization.

4. Using the Calculator

Tips: Enter the absorbance value at 280nm, the extinction coefficient for your protein (often provided in protein databases or literature), and the path length of your cuvette (typically 1cm). All values must be positive numbers.

5. Frequently Asked Questions (FAQ)

Q1: Where can I find extinction coefficients for my protein?
A: Extinction coefficients can be calculated from protein sequence (using tools like ProtParam) or found in literature. They're often expressed as mL/mg/cm or M^-1cm^-1.

Q2: What are typical absorbance values for proteins?
A: For most proteins, A₂₈₀ of 1.0 in a 1cm path length corresponds to ~1mg/mL, but this varies significantly based on aromatic amino acid content.

Q3: What are the limitations of this method?
A: The method is less accurate for proteins with few aromatic amino acids. Nucleic acid contamination can interfere, as they also absorb at 280nm.

Q4: How does path length affect the measurement?
A: Standard cuvettes have 1cm path length. If using different path lengths (e.g., in microvolume spectrophotometers), adjust accordingly.

Q5: Should I blank with buffer?
A: Always blank with the buffer your protein is dissolved in to account for any buffer absorbance at 280nm.