What is A280 and why is it used for protein quantification?

A280 refers to absorbance at 280 nanometers, where aromatic amino acids (tryptophan, tyrosine, and phenylalanine) absorb UV light. This measurement is commonly used for protein quantification because most proteins contain these amino acids in predictable proportions.

What is the typical extinction coefficient for proteins?

Extinction coefficients vary widely between proteins. Common values range from 0.1 to 3.0 mg⁻¹mL⁻¹cm⁻¹. For unknown proteins, a general estimate of 1.0 mg⁻¹mL⁻¹cm⁻¹ is often used, though specific values should be determined experimentally or calculated from amino acid composition.

How does A280 compare to other protein quantification methods?

A280 is fast and non-destructive but can be affected by nucleic acids and other UV-absorbing compounds. Bradford and BCA assays are more sensitive and specific but require reagents and sample consumption. A280 is ideal for pure protein solutions, while colorimetric assays work better for crude samples.

What factors can affect A280 protein concentration measurements?

Several factors can affect accuracy: pH changes, presence of nucleic acids, buffer composition, protein aggregation, and the specific amino acid composition of your protein. Always use appropriate blanks and consider these variables when interpreting results.

Protein Concentration Calculator – mg/mL or A280

How to Use

Method 1: A280 Absorbance

1Measure absorbance at 280nm using a UV-Vis spectrophotometer
2Enter the A280 value and dilution factor
3Input extinction coefficient (1.0 mg⁻¹mL⁻¹cm⁻¹ for unknown proteins)
4Confirm path length (usually 1 cm for standard cuvettes)

Method 2: Mass & Volume

1Enter the total protein mass in milligrams
2Enter the solution volume in milliliters
3Click "Calculate Protein Concentration"

Formula Explanation

Beer-Lambert Law (A280 Method)

A = ε × c × l

c = (A × Dilution Factor) / (ε × l)

Where A is absorbance, ε is extinction coefficient, c is concentration, and l is path length.

Mass/Volume Formula

Concentration = Mass (mg) / Volume (mL)

Direct calculation when protein mass and solution volume are known.

Example Calculation

A280 method:
A280 = 0.8, Dilution factor = 5, Extinction coefficient = 1.2, Path length = 1 cm
Concentration = (0.8 × 5) / (1.2 × 1) = 3.33 mg/mL

Mass/Volume method:
Mass = 25 mg, Volume = 10 mL
Concentration = 25 / 10 = 2.5 mg/mL

Understanding Protein Quantification

Protein concentration determination is essential in biochemistry and molecular biology research. Accurate quantification ensures proper experimental conditions for enzyme assays, structural studies, and protein characterization. The A280 method leverages the natural UV absorption of aromatic amino acids, providing a rapid, non-destructive approach to protein quantification.

The A280 measurement is based on the Beer-Lambert law, where absorbance is directly proportional to concentration. Tryptophan, tyrosine, and phenylalanine residues absorb strongly at 280nm, making this wavelength ideal for protein detection. However, the extinction coefficient varies significantly between proteins depending on their amino acid composition.

Compared to colorimetric methods like Bradford and BCA assays, A280 measurements are faster and preserve sample integrity. Bradford assays use Coomassie dye binding and are sensitive to protein structure, while BCA assays rely on copper reduction and can be affected by reducing agents. Each method has specific advantages depending on sample purity and experimental requirements.

Protein Quantification Methods Comparison

Method	Speed	Sensitivity	Sample Required	Best For
A280	Very Fast	Moderate	Non-destructive	Pure proteins
Bradford	Fast	High	Small volume	Crude samples
BCA	Moderate	Very High	Small volume	Low concentrations
Lowry	Slow	High	Moderate volume	Research standard

Chart: Comparison of common protein quantification methods showing their relative strengths and ideal applications.

A280 Spectrophotometry Technique

Diagram: A280 spectrophotometry setup showing UV light at 280nm passing through protein sample for concentration measurement.

Key Components:

• UV light source (280nm)
• Sample cuvette (1cm path length)
• Detector/photometer
• Reference blank

Measurement Process:

• Blank with buffer solution
• Measure sample absorbance
• Apply Beer-Lambert law
• Calculate concentration

Applications in Protein Research

Protein concentration measurement is fundamental to biochemical research, from enzyme kinetics studies to structural biology preparations. Accurate quantification ensures reproducible results in protein purification, crystallization, and functional assays. The A280 method is particularly valuable for monitoring protein elution during chromatography and assessing purification efficiency.

Modern proteomic workflows require precise concentration measurements for downstream applications including mass spectrometry, Western blotting, and enzymatic assays. The choice of quantification method depends on sample purity, concentration range, and experimental requirements. A280 measurements excel in real-time monitoring applications where sample preservation is critical.

For comprehensive molecular biology calculations, explore our related tools including DNA concentration calculator and DNA ligation calculator for complete laboratory workflow support.

Protein ConcentrationCalculator