Bradford assay

When Coomassie brilliant blue G-250 binds to proteins, the absorption maximum of the color changes (465 nm without protein 595 nm with protein). The increase of the absorption to 595 nm is a measure of the solution s protein concentration. 

Advantages The color is completely developed after 2 minutes, the coloring varies little between different proteins, and there is only minimal pipetting work. The reagents are available via retail (e.g., Bio-Rad and Pierce). 

Problems The reaction takes place in an acidic environment in which many proteins fall out of solution. Strong lyes and commonly used detergents such as TRlTON-X-100, SDS (sodium dodecylsulfate), or CHAPS interfere. 

Bradford, M. (1976). A Rapid and Sensitive Method for the Quantitation of Microgram Quantities of Protein Utilizing the Principles of Protein-dye Binding, Anal. Biochem. 72 248-254. 

and Northcote, D. (1981). Minimization of Variation in the Response to Different Proteins of the Coomassie Blue G Dye-binding Assay for Protein, Anal. Biochem. 116 53-64. 

---

For quantitative analysis of protein concentration the colorimetric Bradford-assay [147] is most commonly used. Here another Coomassie dye, Brilliant Blue G-250, binds in acidic solutions to basic and aromatic side chains of proteins. Binding is detected via a shift in the absorption maximum of the dye from 465 nm to 595 nm. Mostly calibration is performed with standard proteins like bovine serum albumin (BSA). Due to the varying contents of basic and aromatic side chains in proteins, systematic errors in the quantification of proteins may occur. 

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.)... 

Protein concentrations were determined by performing Bradford assay using bovine serum albumin as a standard. Equal amounts... 

The abundance and ease of purification made bovine serum albumin (BSA) an early standard in protein chemistry, and BSA is widely used as protein standard in biuret, Lowry, and Bradford assays as well as a molecular weight... 

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. 

Basic Protocol 4 The Coomassie Dye-Binding (Bradford) Assay for... 

This unit describes four of the most commonly used total protein assay methods. Three of the four are copper-based assays to quantitate total protein the Lowry method (see Basic Protocol 1 and Alternate Protocols 1 and 2), the bicinchoninic acid assay (BCA see Basic Protocol 2 and Alternate Protocols 3 and 4), and the biuret method (see Basic Protocol 3 and Alternate Protocol 5). The fourth is the Coomassie dye binding or Bradford assay (see Basic Protocol 4 and Alternate Protocols 6 and 7), which is included as a simple and sensitive assay, although it sometimes gives a variable response depending on how well or how poorly the protein binds the dye in acidic pH. A protein assay method should be chosen based on the sensitivity and accuracy of method as well as the condition of the sample to be analyzed. 

THE COOMASSIE DYE-BINDING (BRADFORD) ASSAY FOR DETERMINING TOTAL PROTEIN... 

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. 

Determine the protein concentration of the soluble protein extracts by Bradford assay (20) to confirm that effective cell lysis has occurred see Note 5). 

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. 

We generally assess the protein concentration of new chemokine stock solutions by OD280 using the appropriate extinction coefficient for each chemokine or by Bradford assay. Although this is not often practical when very small quantities of chemokine are purchased because 10-20% of the material may be sacrificed for an accurate determination, we have found that the actual protein concentration may vary by as much as 2CM-0% between lots which can significantly effect the reproducibility of chemotaxis assays. 

Dye-Binding (Bradford) Assay. The binding of proteins to Coomassie Brilliant Blue 250 causes a shift in the absorbance maximum of the dye from 465 nm to an intense band at 595 nm. Determination of the increase in absorbance at 595 nm as a function of protein added provides a sensitive assay... 

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. 

Figure 5-1. Standard curve for Bradford assay. tion prepared either by weight or, for a protein of The standard curve is generated by dilution of known extinction coefficient, from accurate mea-the stock protein solution of known concentra- surement of absorbance.

---