The Bradford Method for Protein Quantitation （布拉德福德蛋白质定量方法）.pdf

The Bradford Method 15 4 The Bradford Method for Protein Quantitation Nicholas J. Kruger 1. Introduction A rapid and accurate method for the estimation of protein concentration is essential in many fields of protein study. An assay originally described by Bradford (1) has become the preferred method for quantifying protein in many laboratories. This tech- nique is simpler, faster, and more sensitive than the Lowry method. Moreover, when compared with the Lowry method, it is subject to less interference by common rea- gents and nonprotein components of biological samples (see Note 1). The Bradford assay relies on the binding of the dye Coomassie Blue G250 to pro- tein. Detailed studies indicate that the free dye can exist in four different ionic forms for which the pKa values are 1.15, 1.82, and 12.4 (2). Of the three charged forms of the dye that predominate in the acidic assay reagent solution, the more cationic red and green forms have absorbance maxima at 470 nm and 650 nm, respectively. In contrast, the more anionic blue form of the dye, which binds to protein, has an absorbance maxi- mum at 590 nm. Thus, the quantity of protein can be estimated by determining the amount of dye in the blue ionic form. This is usually achieved by measuring the absor- bance of the solution at 595 nm (see Note 2). The dye appears to bind most readily to arginyl and lysyl residues of proteins (3,4) . This specificity can lead to variation in the response of the assay to different proteins, which is the main drawback of the method (see Note 3). The original Bradford assay shows large variation in response between different proteins (5–7). Several modifica- tions to the method have been developed to overcome this problem (see Note 4). How- ever, these changes generally result in a less robust assay that is often more susceptible to interference by other chemical