Shifting Focus in the Bradford Assay: Interfering Compounds Re-Examined

Since its introduction in 1976, the Bradford assay has served as a gold standard for protein quantification across a wide range of applications. While its limitations—including protein-to-protein variation in dye binding, challenges in selecting a representative calibration standard, and susceptibility to matrix interferences—are recognized, the relevant information remains scattered throughout the literature, with little quantitative guidance available for assay optimization. Here, we review interfering compounds reported in the literature during nearly 50 years and report a systematic characterization of a panel of potential interfering compounds, evaluating the effects of 29 different substances in the presence or absence of the protein analytes. Our findings revealed that 12 of the tested compounds induce significant artefacts in the Bradford assay, with minimal interfering concentrations varying widely across compounds. Detergents were confirmed as the most problematic interference; furthermore, two novel groups of interfering compounds were identified, represented by the transfection reagents and oligoarginine peptides with molecular weight below 3 kDa. Importantly, the resulting artefacts were also observed in complex biological matrices. While these compounds also affected the Lowry assay, the magnitude of the artefacts was substantially lower than that observed in the Bradford assay. This study will provide a valuable resource for researchers working in proteomics and related fields, offering practical insights for improving the reliability of Bradford-based protein quantification.