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Protein Concentration Equation:
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The A280 method estimates protein concentration based on ultraviolet absorbance at 280nm. This method relies on the absorbance properties of aromatic amino acids (tryptophan and tyrosine) in proteins, providing a quick and non-destructive way to measure protein concentration.
The calculator uses the simplified A280 equation:
Where:
Explanation: This simplified equation provides a quick estimate of protein concentration based on the average extinction coefficient for most proteins.
Details: Accurate protein concentration measurement is essential for various biochemical applications including protein purification, enzyme kinetics studies, Western blotting, and protein quantification for experimental setups.
Tips: Enter the absorbance reading at 280nm. Ensure the spectrophotometer is properly calibrated and the sample is appropriately diluted to fall within the linear range of the instrument (typically 0.1-1.0 AU).
Q1: Why use 0.96 as the conversion factor?
A: 0.96 is a general conversion factor that works well for most proteins. However, for precise measurements, protein-specific extinction coefficients should be used.
Q2: What are the limitations of A280 measurement?
A: This method can be affected by nucleic acid contamination, buffer components that absorb at 280nm, and variations in aromatic amino acid content between different proteins.
Q3: When should I use this method?
A: A280 is ideal for quick estimates and purified protein samples. For crude extracts or complex mixtures, other methods like Bradford or BCA assays may be more appropriate.
Q4: How accurate is this estimation?
A: The accuracy depends on the protein's specific aromatic amino acid content. For most standard proteins, the error is within 10-20% of the true concentration.
Q5: Can I use this for all protein types?
A: While generally applicable, proteins with unusual aromatic amino acid content (very high or very low) may require specific extinction coefficients for accurate measurement.