Bradford Protein Assay

The Bradford assay quantifies protein concentration by measuring the color change of a dye, Coomassie Brilliant Blue G-250, from brown to blue.

Bradford protein assays are colorimetric tests that determine the total amount of protein in solution against a known standard curve, usually bovine serum albumin (BSA). The assay principle relies upon the absorbance shift of the Bradford reagent, Coomassie Brilliant Blue G-250, when bound to proteins. Under acidic conditions, the free dye is in its cationic form, which appears reddish-brown, with a maximum absorbance at 470 nm. In the presence of proteins, it changes to its anionic form, as strong, noncovalent dye-protein complexes are created, resulting in a color change of the dye to blue, with a maximum absorbance at 595 nm. The degree of color change, as measured by optical density (OD), or absorbance, at 595 nm is directly proportional to the protein concentration, with higher absorbance values corresponding to greater total protein in solution.

The Bradford method of protein quantification has some advantages over alternative methods, such as absorbance measurements at 280 nm. First, it utilizes readings in the visible spectrum range (595 nm). This allows for experiments to be conducted when UV spectrophotometers are unavailable. Additionally, the color change in the Bradford reagent is apparent by simple visual inspection.

Compared to 280 nm quantification methods, Bradford assays are susceptible to different biases stemming from protein composition. That is, measurements of absorbance at 280 nm relies on the target protein containing side chains with aromatic rings, such as tryptophan, tyrosine and phenylalanine. On the other hand, the Van der Waals forces and hydrophobic interactions, which lead to the dye-protein complexes of the Bradford method, depend upon the presence of basic amino acids, such as arginine, lysine and histidine. Proteins which do not contain these side chains may invalidate each test method, respectively.

One common limitation of the Bradford protein assay is its small dynamic range across which the method is linear. This is usually between 0 µg / mL to 10 µg / mL, beyond which measured dye OD no longer increases proportionally with protein concentration. The reason for this is due to the overlapping spectrums of the two forms of the Coomassie Brilliant Blue dye. One solution to this problem is to use the ratio of absorbances at 595 nm and 460 nm. This tends to yield a 10-fold increase in the dynamic range of the assay.

Bradford, M. M. (1976). A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. Analytical biochemistry, 72(1-2), 248-254.

Provided as reference. AAT Bioquest's Bradford reagent formulation is optimized for improved performance.

1. Dissolve 100 mg of Coomassie Brilliant Blue G-250 in 50 mL of 95% ethanol.
2. Add 100 mL of 85% (w/v) phosphoric acid. Stir continuously until the dye has completely dissolved.
3. Dilute to 1 liter with water.
4. Filter with Whatman® Grade 1 filter paper.

Noble, J. E., & Bailey, M. J. (2009). Quantitation of protein. Methods in enzymology, 463, 73-95.