Interferences of suspended clay fraction in protein quantitation by several determination methods

Seven current methods of protein quantitation, Bradford (standard, micro, and 590/450 nm ratio), Lowry, bicinchoninic acid (BCA), UV spectrophotometry at 280 nm, and Quant-iT fluorescence-based determination, were compared with regard to their susceptibility to interferences due to the presence of suspended and not easily detectable clay particles. Bovine serum albumin (BSA) and Na-Wyoming montmorillonite were selected as model protein and reference clay, respectively. Protein-clay suspension mix­tures were freshly prepared for each assay to simulate supernatants not completely centrifuged in batch sorption/kinetic ex­periments. Seven fix­ed increasing levels of clay (0.0, 0.00725, 0.0145, 0.029, 0.058, 0.145, 0.435 mg ml¡1) were mix­ed with diVerent levels of BSA in an appropriate range for each assay. To ascertain the interfering eVect of diVerent levels of clay, the theoretical concentrations of BSA were plotted against the estimated BSA concentrations of the samples, as obtained from the calibration curve of each method. A correct quantitation of the BSA concentration not influenced by clay would be described by a regression line with slope (b) not significantly diVerent from 1 and an intercept (a) not signif- icantly diVerent from zero. At the lowest clay levels (0.00725 mg ml¡1) a significant interference was evident for Bradford micro, Bradford 590/450, UV, and fluorescence. The three methods (Bradford standard, Lowry, and BCA) that seemed to show the better performances in the presence of clay after this first screening step also underwent an ANCOVA analysis, with the measured BSA concentrations as depen- dent variable and the clay concentrations as covariate. The Bradford standard and BCA methods were aVected by a clay-dependent inter- ference on BSA quantitation. The Lowry assay was the only method that gave correct estimates of BSA concentrations in the presence of any of the clay levels tested.