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 – from agricultural to clinical samples. While its limitations – including the necessity of a calibration curve, challenges in selecting a representative protein 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 perform an exhaustive review of the interfering compounds reported in 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 notable interfering compounds were identified, represented by the transfection reagents and oligoarginine peptides with molecular weight below 3 kDa. Principal component analysis (PCA) of the Coomassie Brilliant Blue G-250 absorbance spectra allowed grouping of interfering compounds based on the nature of the spectral distortions. We anticipate that 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.