Understanding A280 Protein Concentration
A280 measurement is a fast spectrophotometric method for estimating protein concentration. It reads ultraviolet absorbance near 280 nm. Aromatic amino acids absorb strongly in this region. Tryptophan contributes the most signal. Tyrosine adds a smaller signal. Disulfide cystine adds a small part. Because each protein has a different sequence, its extinction coefficient also differs.
Why Blank Correction Matters
A raw absorbance value is rarely perfect. Buffer, salts, detergents, and cuvette marks can add background signal. A blank correction removes this background. A320 correction helps when light scattering is present. This is useful for turbid samples. Dilution correction then scales the measured value back to the original tube. Path length correction adjusts readings from short path plates.
Choosing the Right Method
The molar extinction method is best when you know molecular weight and molar extinction coefficient. It returns molarity and mass concentration. The mass extinction method is useful when a supplier gives absorbance for one milligram per milliliter. Sequence mode estimates extinction from tryptophan, tyrosine, and cystine counts. Nucleic acid correction is included for mixed samples, but it is only approximate.
Interpreting the Result
A clean protein sample usually has a stable A260/A280 ratio. A high A260 value suggests nucleic acid contamination. A high A320 value suggests particles or bubbles. Very high A280 readings may exceed the linear range. Dilute the sample and measure again. Repeat readings improve confidence. Always use the same buffer as the blank.
Good Laboratory Practice
Wipe cuvettes before reading. Avoid fingerprints and foam. Mix samples gently. Record dilution factors clearly. Use matched cuvettes when possible. For microplates, confirm the optical path length. Use calibrated instruments. Keep extinction coefficients with sample records. The final concentration should be reported with units, method, path length, and correction details. This makes the result traceable. It also helps another scientist repeat the calculation. The calculator supports these records by showing corrected absorbance, concentration, molarity, ratios, warnings, and export files.
Limits and Safety Notes
A280 is not a purity assay by itself. Some buffers contain absorbing ingredients. Reducing agents may affect baseline quality. Aggregates can scatter light. Use colorimetric assays when sequences are unknown. Use chromatography data when purity is critical. Treat every output as an analytical estimate.