The Bradford Assay

Fig. 7. Coomassie Brilliant Blue R-250 (I) is used to stain proteins, e.g., after gel-electrophoretic separation, its derivative G-250 (II) is applied in the Bradford assay for protein quantification... 

The first assay to be employed for protein concentration is the Bradford assay, a commercially available colorimetric assay used to quantitate the total extracted protein. Amb a 1 is approximately 1% of the total protein extracted from ragweed pollen hence the Bradford assay does not reflect Amb a 1 concentration. However, at this step of the production process, the protein concentration is used to calculate final yields and not to make time-dependent or expensive decisions. Hence the nonspecific Bradford assay is ideal. A simpler direct absorbance method is not suitable due to the presence of a nonprotein chromophore in the ragweed extract. 

The actual Amb a 1 concentration of the extract can be quantitated using a reversed-phase HPLC method developed at Dynavax. This is a custom two-step method that employs chromatography to separate the Amb a 1 from the other extracted proteins. The Amb a 1 concentration is then determined from the resolved Amb a 1 peak area and a standard curve of purified Amb a 1. This is the only step at which the Amb a 1 concentration of the process material is measured by a two-step process. Following the extraction step, the Amb a 1 rapidly becomes enriched over two purification steps, and the Bradford assay adequately reflects Amb a 1 concentration through the remainder of the process. 

Protein concentration was determined using the Bradford assay at 595 nm. 100 pL of the sample were introduced into a cuvette containing 5 mL of Bradford solution (100 mg of Coomassie blue, 50 mL of ethanol and 100 mL of 85 % phosphoric acid dissolved in 850 mL of H2O). The solutions were incubated for 5 min at room temperature. The absorbance was measured at 595 nm. The protein concentration in the sample was determined using a calibration curve plotted with serum albumin (1 mg mL ) as a standard.)... 

Standard calibration curve for the Bradford assay using bovine gamma globulin as standard protein. 

B 5. Below is a table prepared by a biochemistry student to construct a standard curve for protein analysis. The Bradford assay was used with bovine serum albumin (BSA, 0.1 mg/mL) as standard protein. Complete the table by filling in the weight of BSA in each tube and the approximate A595 that will be obtained for each tube. Assume the procedure was conducted correctly. 

Example 4 The Bradford protein assay is one of the most used spec-trophotometric assays in biochemistry. (For a discussion of the Bradford assay, see Chapter 2.) Solutions of varying amounts of a standard protein are mixed with reagents that cause the development of a color. The amount of color produced depends on the amount of protein present. The absorbance at 595 nm of each reaction mixture is plotted against the known protein concentration. A protein sample of unknown concentration is treated with the Bradford reagents and the color is allowed to develop. 

Compare these results with the results from the Bradford assay. Explain any differences. 

The most frequently used protein assay is based on a method after Bradford (Bradford, 1976), which combines a fast and easily performed procedure with reliable results. However, the Bradford assay has sensitivity limitations and its accuracy depends on comparison of the protein to be analyzed with a standard curve using a protein of known concentration, commonly bovine serum albumin (BSA). Many commercially available protein assays such as those from Pierce or BioRad rely on the Bradford method. The assay is based on the immediate absorbance shift from 465 nm (brownish-green) to 595 nm (blue) that occurs when the dye Coomassie Brilliant Blue G-250 binds to proteins in an acidic solution. Coomassie dye-based assays are known for their non-linear response over a wide range of protein concentrations, requiring comparison with a standard. The dye is assumed to bind to protein via an electrostatic attraction of the dye s sulfonic groups, principally to arginine, histidine, and lysine residues. It also binds weakly to the aromatic amino acids, tyrosine, tryptophan, and phenylalanine via van der Waals forces and hydrophobic interactions. 

For expression of BCCP-p53 fusion proteins, we typically found the protein concentration in crude lysates to be 5 mg/mL, and we estimated that BCCP-p53 was present at approx 1% of total soluble protein. When expressing a number of clones in parallel for array fabrication, the Bradford assay can conveniently be done on all clones in parallel using a microtiter plate format. However, it would be laborious to carry out SDS-PAGE analysis on all clones, so typically we assess only a selection of clones in this way, since the absolute expression level is not critical for array fabrication. 

Tris buffers Tris is also a much used buffer. However, it has one great disadvantage its pH is highly dependent on temperature and concentration. The pH of a Tris buffer will increase from 8.0 at 25 °C to 8.6 on cooling to 5 °C and on dilution of a 0.1 M solution at pH 8.0 to 0.01 M, the pH will fall to 7.9. This problem can only really be avoided by adjusting the pH of the buffer under the conditions of temperature and concentration where it is to be used. In addition, Tris has been shown, like phosphate discussed above, to interfere with many enzymic reactions, particularly those which have aldehyde intermediates. It also interferes with many chemical reactions, like the coupling of proteins to activated surfaces, and the Bradford assay for spectrophotometric determination of proteins. 

The enzyme activity was measured by a continuous spectrophotometric assay (see Methods), active site concentration was determined by FAD absorption at 452 nm (8 = 12.83 mM-icm-i) as described by Frederick et al. (1990) and the protein concentration was measured by the Bradford assay (BioRad reagent) using bovine serum albumin as standard, or by its absorption at 280 nm using a published factor of 1.67 O.D. per mg (Swoboda Massey, 1965). The specific activity was 430 U/mg, and the overdl yield of enzyme, based on active sites measurement (452 nm absorption), was about 40%. 

To compare activity between samples, all are normalized for total cytosolic protein. We carry this out using the BioRad protein assay (a commercial preparation of the Bradford assay), and a standard curve from BSA protein standard... 

The Bradford assay is particularly suited to samples where there is a high lipid content which may interfere with the Lowry assay. 

Total protein in an unknown sample was estimated using the Lowry and Bradford assays. Results were 33 2 pg/mL from the Lowry assay and 21 1 pg/mL from the Bradford assay, using stock solutions of BSA as standards in each assay. The unknown sample was then thoroughly oxygenated, and the assays were repeated. The Lowry method yielded 22 1 pg/mL, while the Bradford results did not change. Why did the results of the Lowry assay decrease by 33% ... 

Collect the supernatants and measure the protein concentration in each sample using the Bradford assay kit (5 x 10 Jurkat cells have a total amount of -250 pg protein). 

Use lysis buffer if needed to adjust for equal loading among the samples according to the protein concentrations determined in the Bradford assay. 

Another assay, the Bradford assay, also known as the Coomassie dye binding method, was first described in 1976 [19]. In an acidic environment, proteins will bind to Coomassie dye and cause a shift from the reddish brown color (465 nm) to the blue dye protein complex read at 595 nm. The development of the color is attributed to the presence of the basic amino acids arginine, lysine, and histidine. Van der Waals forces and hydrophobic interactions account for the dye binding and the number of Coomassie blue dye molecules bound is roughly proportional to the number of positive charges on the molecule. A protein molecular weight of about 3 kDA is required for successful color development. The Bradford assay dose response is nonlinear and this method demonstrates the greatest difference in reactivity with BSA compared to BGG. 

The protein content of the commercial enzyme preparations and the fungal supernatant samples were determined using the Bradford assay [23]. 

Various colorimetric methods are also available, based on non-specific dye binding to polypeptide chains, one of the more common being the Bradford assay. One drawback with such methods is that the actual colour intensity (absorbance) developed is not absolute, but depends on the specific protein. Calibration can therefore be a problem if accurate concentrations are required. 

Furthermore, every protein determination is sensitive to detergents or certain ions. Hence, when presenting a concentration value it is good practice to also mention the assay that was used as well as the benchmark protein. The methods of choice are the Bradford assay and the BCA (bicinchoninine acid) assay. Anyone working with membrane proteins and detergents should use the BCA assay. Otherwise, the choice between the BCA and the Bradford assays seems to be a question of taste. 

Determine the protein concentration of the soluble, crude protein extract using the Bradford assay (25) to confirm that effective cell lysis has occurred (see Note 4). 

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