Systematic comparisons of various spectrophotometric and colorimetric methods to measure concentrations of protein, peptide and amino acid: Detectable limits, linear dynamic ranges, interferences, practicality and unit costs

There is limited and inconclusive information regarding detectable limits and linear dynamic ranges of various quantitative protein assays. We thus performed systematic comparisons of seven commonly used methods, including direct spectrophotometric quantitation at λ205 and λ280 nm (A205 and A280, respectively), bicinchoninic acid (BCA), Biuret, Bradford, Lowry and Ninhydrin methods. Purified BSA, porcine kidney extract, tryptic digested peptides derived from purified BSA, and glycine, were used as representative purified protein, complex protein mixture, peptide and amino acid, respectively. Bradford method was the most sensitive assay (LOD=0.006 mg/ml) and had the widest range of detectability (LOD–UOD=0.006–100 mg/ml) for purified protein and complex protein mixture. For peptide, A205, A280, Lowry and Ninhydrin methods had a comparable LOD (0.006 mg/ml), but Ninhydrin method had the widest detectability range (LOD–UOD=0.006–100 mg/ml). For amino acid, A205 and Ninhydrin methods had a comparable LOD (0.006 mg/ml), but A205 had a wider detectability range (LOD–UOD=0.006–6.250 mg/ml). Biuret method offered the widest linear dynamic range for purified protein and complex protein mixture (0.391–100 mg/ml), A280 offered the widest linear dynamic range for peptide (0.024–6.250 mg/ml), and Ninhydrin method offered the widest linear dynamic range for amino acid (0.024–0.195 mg/ml). Both Laemmliʹs and 2-D lysis buffers had dramatic interfering effects on all assays. Concerning the practicality and unit costs, A205 and A280 were the most favorable. Among the colorimetric methods, Bradford method consumed the least amount of samples and shortest analytical time with the lowest unit cost. These are the most extensive comparative data of commonly used quantitative protein assays that will be useful for selecting the most suitable method for each study.