Understanding A280 Protein Measurements
A280 testing estimates protein concentration from ultraviolet absorbance. It works because tryptophan and tyrosine absorb strongly near 280 nm. Cystine also contributes a smaller signal. The result depends on the protein sequence. A pure protein with a known extinction coefficient gives the best estimate. Mixed lysates need more caution.
Why Inputs Matter
A blank removes buffer absorbance. Path length adjusts short cuvettes and microplate readings. Dilution returns the measured value to the original sample. Turbidity correction subtracts A320, which helps when particles scatter light. The A260 value helps flag nucleic acid contamination. A high A260/A280 ratio suggests DNA or RNA may be present.
Choosing a Method
The molar extinction method is best for a purified protein. Enter molecular weight and molar extinction coefficient. The calculator converts molar concentration into mg/mL. The mass extinction method is useful when the coefficient is already stated as mL/mg/cm. The E1% method is common in product sheets. It means the absorbance of a one percent solution in a one cm path.
Practical Lab Notes
Keep absorbance inside the linear range of the instrument. Many spectrophotometers work best below 1.5 absorbance units. Dilute samples that read too high. Mix samples well before reading. Use matched cuvettes when possible. For microplates, confirm the path length correction. Bubbles can cause serious error. Wipe cuvettes before measurement.
Interpreting Results
The main result is concentration in mg/mL. The same number also equals g/L. The tool also reports µg/mL. If molecular weight is known, it reports µM. Sample mass is estimated from the entered volume. This helps prepare loading amounts for gels or assays.
Good Reporting Practice
Record the method used with every result. Include blank, dilution factor, path length, and correction choice. Note the extinction coefficient source. A280 is fast and nondestructive. It is not always specific. Confirm important samples with another assay when contaminants, detergents, or unusual buffers are present.
Common Limitations
Some proteins contain few aromatic residues. Their A280 signal can be weak. Other proteins contain many aromatic residues. Their signal can be strong at low mass. Reducing agents, nucleotides, imidazole, and dirty plastic can shift readings. Always inspect spectra when accuracy matters. A smooth peak near 280 nm supports cleaner measurement and confidence.