Why might protein measurements become inaccurate when proteins undergo denaturation?

When a protein unfolds, its three‑dimensional shape is lost and amino acids that were buried inside become exposed. This exposes new sites that can bind dyes or reagents used in common assays, such as the Coomassie dye in Bradford or the copper ions in a BCA test, so the signal no longer reflects the true amount of protein. The unfolded protein may also scatter light differently or absorb at 280 nm more strongly because aromatic residues are more exposed, causing the measured absorbance to change. Consequently, the assay’s calibration curve, which assumes native structure, becomes inaccurate. For example, a denatured protein can give a Bradford reading that is 30 % higher than its actual concentration because the dye binds more strongly to the newly exposed lysines.