ANALYTICAL BIOCHEMISTRY 236, 302±308 (1996) ARTICLE NO. 0171 Linearization of the Bradford Protein Assay Increases Its Sensitivity: Theoretical and Experimental Studies

struction of the calibration graph. This nonlinearity Determination of microgram quantities of protein in presents a serious problem outside this narrow the Bradford Coomassie brilliant blue assay is accom- range of protein concentrations and when microgram plished by measurement of absorbance at 590 nm. amounts of protein are not available. Over 100 works However, an intrinsic nonlinearity compromises the have attempted to improve the Bradford assay (for sensitivity and accuracy of this method. It is shown review, see (2)). Some have addressed the nonlinear-that under standard assay conditions, the ratio of the ity problem (3±5), but all offered only a partial solu-absorbances, 590 nm over 450 nm, is strictly linear with tion. protein concentration. This simple procedure in- Three charge forms of the Coomassie brilliant blue creases the accuracy and improves the sensitivity of dye are present in equilibrium at the usual acidic the assay about 10-fold, permitting quantitation down pH of the assay. The red, blue, and green forms haveto 50 ng of bovine serum albumin. Furthermore, pro-absorbance maxima at 470, 590, and 650 nm, respec-tein assay in presence of up to 35-fold weight excess of tively (6). The blue is the form that binds the protein,sodium dodecyl sulfate (detergent) over bovine serum forming a complex that intensely absorbs light at 594albumin (protein) can be performed. A linear equation nm (7, 8). Therefore one may monitor the decrease inthat perfectly ®ts the experimental data is provide