1. What is the 280 nm Absorbance Method?

The 280 nm absorbance method estimates protein concentration by measuring the absorption of ultraviolet light at 280 nm, where tryptophan and tyrosine residues absorb strongly. This quick and non-destructive method is widely used in biochemistry labs.

2. How Does the Calculator Work?

The calculator uses the Beer-Lambert law:

Where:

• \( C \) — Protein concentration (mg/mL)
• \( A_{280} \) — Absorbance at 280 nm (AU)
• \( \epsilon \) — 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 experiments requiring precise protein amounts, such as enzyme assays, western blotting, and protein purification.

4. Using the Calculator

Tips:

• Measure absorbance at 280 nm using a spectrophotometer
• Use the protein's extinction coefficient (often provided in protein databases)
• Standard cuvettes typically have 1 cm path length
• For diluted samples, multiply result by dilution factor

5. Frequently Asked Questions (FAQ)

Q1: How do I find my protein's extinction coefficient?
A: Check protein databases (like UniProt) or calculate it from amino acid sequence using tools like ProtParam.

Q2: What's the typical range for extinction coefficients?
A: Most proteins have ε values between 0.1-3.0 mL/(mg·cm). Antibodies often have higher values (~1.4).

Q3: Why might my 280 nm measurement be inaccurate?
A: Nucleic acid contamination (absorbs at 260 nm), turbidity, or buffers that absorb at 280 nm can interfere.

Q4: What if I don't know my protein's extinction coefficient?
A: Use a standard curve with BSA or another protein standard, or consider alternative methods like Bradford assay.

Q5: What's a typical concentration range for this method?
A: Best for 0.1-2 mg/mL. For very dilute samples, consider using a shorter wavelength (205 nm) or concentration methods.