Linearization of the Bradford Protein Assay

Abstract

Determination of microgram quantities of protein in the Bradford Coomassie brilliant blue assay is accomplished by measurement of absorbance at 590 nm. This most common assay enables rapid and simple protein quantification in cell lysates, cellular fractions, or recombinant protein samples, for the purpose of normalization of biochemical measurements. However, an intrinsic nonlinearity compromises the sensitivity and accuracy of this method. It is shown that under standard assay conditions, the ratio of the absorbance measurements at 590 nm and 450 nm is strictly linear with protein concentration. This simple procedure increases the accuracy and improves the sensitivity of the assay about 10-fold, permitting quantification down to 50 ng of bovine serum albumin. Furthermore, the interference commonly introduced by detergents that are used to create the cell lysates is greatly reduced by the new protocol. A linear equation developed on the basis of mass action and Beer's law perfectly fits the experimental data. Protocol Linearization of the Bradford Protein Calibration Graph:The Coomassie brilliant blue protein assay, commonly known as the Bradford assay 1 , is widely used because of its rapid and convenient protocol as well as its relative sensitivity. Unfortunately, there is a large degree of curvature over a broad range of protein concentrations (Fig. 1). Therefore, only a narrow range of relatively high protein concentrations, 2-10 mg/ml BSA, is used for the calibration graph, which then better fits linear regression (Fig. 1, green). However, the nonlinearity requires protein concentration of the unknown samples to fall within the limited range of the calibration graph in order to avoid a large error, and it also reduces the accuracy within the limited range. The nonlinearity presents a serious problem in particular when microgram amounts of protein are not available, and it often requires multiple dilutions of the unknown samples.As noted in the original Bradford paper, "the source of the nonlinearity is in the reagent itself since there is an overlap in the spectrum of the two different color forms of the dye." 1 . In fact, three forms of the Coomassie brilliant blue dye are in acid-base equilibrium at the usual acidic pH of the assay 2 . The red, blue, and green forms have absorbance maxima at 470, 590, and 650 nm, respectively (Fig. 2). The blue is the form that binds the protein, forming a complex that intensely absorbs light at 594 nm 3, 4 (Fig. 2). Bradford also noted that "the background value for the reagent is continually decreasing as more dye is bound to protein" 1 (Fig. 3). Therefore, we attempted to calculate the reduction of the 590 nm background as increasing protein quantities are added, by measuring the change of absorbance at 450 nm, where the protein-dye complex does not absorb. We found that the decreasing background partially, but not fully, accounts for the nonlinearity (Fig. 4).We then hypothesized that the decrease in free dye concentration produces another distortion of the l...

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