Interference study should be performed for every protein measurement method used

It is particularly important, when analyzing biological material, for the m easurement procedure to be specific to the analyte and not to suffer interf erence by the matrix effect. Tissue fraction studies also require rapid and accurate methods to estimate the concentration of protein in solutions as well as many measurement methods used in medical laboratories. The design o f this study is based on a comparison of the Lowry and the bicinchoninic ac id (BCA) methods for the measurement of the total protein concentrations of rat liver subcellular fractions. In our experiment, subcellular fractions enriched in peroxisomes (POs) obtained by differential centrifugation were then further separated by means of density gradient centrifugation. We perf ormed the protein measurement assays on all fractions obtained during the p urification steps. The protein contents of the fractions obtained were dete rmined by the two methods. The method comparison statistics were performed by linear Deming regression analysis and Altman and Bland bias plot. The re gression equation was unacceptable, indicating that the last three fraction s separated by means of Nycodenz discontinuous density gradient centrifugat ion gave remarkably divergent results. For the Lowry method, the Nycodenz e ffect could not be eliminated with the use of interference blank. In additi on to Nycodenz, the potentially interfering compound used in the isolation procedure as isolation medium was 3-(morpholino)propane sulfonic acid (MOPS ). In decreased concentrations of MOPS (10 mM), interference blank should b e used for correct measurement with Lowry, but in practical use 10 mM does not provide buffering potency. In the BCA method, interference blank correc tion seemed to eliminate the measurement error in all concentrations of Nyc odenz. There was no MOPS effect on the BCA measurement assay. Referring to deviations as sample-inherent matrix effects, we concluded that not only on e, but more measurement methods should be used in order to make a correct p rotein measurement.