Bradford Assay


Bio-Rad's Bradford assays provide a simple and accurate method for determining protein concentrations. The binding of the Bradford reagent to proteins results in a color change which is measured with a spectrophotometer or a microplate reader. Choose the kit that meets your needs.

The Bradford Assay: Colorimetric Protein Determination with Coomassie Blue

The Bradford assay is based on the use of the dye Coomassie Brilliant Blue G-250, which is frequently abbreviated as Coomassie G-250 or Coomassie Blue. This is one of two Coomassie dyes that are often confused. Coomassie R-250 is used to stain protein gels but is not used in protein assays. In addition to being used in the assay, Coomassie G-250 can also be used to stain protein gels, although it is less sensitive than Coomassie R-250.

Under acidic conditions, Coomassie G-250 is cationic, mainly doubly protonated, and is red, whereas in neutral conditions the dye is green, and the anionic form is blue. The Bradford reagent is an acidified solution of Coomassie G-250; the dye is thus primarily protonated and red.

The basis for the assay is that in order for the Coomassie dye to bind stably to protein, it needs to be doubly protonated. When the dye comes in contact with protein, the first electron is donated to charged groups on the protein. This disrupts the structure of the protein, resulting in exposure of hydrophobic pockets. The dye binds to these pockets, with the sulfonic acid groups binding to positive amines. In addition, there is attraction due to Van der Waals forces. The stably bound Coomassie G-250 is the blue, unprotonated form.

Protein Dye Binding in Bradford Assays

The change in the color of Coomassie G-250 from red to blue upon binding protein is measured spectroscopically. In the absence of protein, when the dye is red, Bradford reagent has an absorbance maximum (Amax) of 470 nm. In the presence of protein, the change to the anionic blue form of the dye shifts the Amax to 595 nm.

Since the amount of the blue anionic form is proportional to the amount of protein in the sample, the quantity of protein in a sample can measured directly by measuring the absorption at 595 nm. The neutral green form of Coomassie G-250 that has donated an electron but is not bound to protein does not interfere with measurement in the assay since it has an Amax of 650 nm.

Bradford Assay Standards

The assay uses standards to both quantify the amount of protein in samples and to subtract any background due to interfering substances that can shift the ratios between the three forms of the dye.

The concentration range of standards in the kits cover the linear range of the assay. Since the curve flattens at high concentrations of dye, the amount of protein in the sample will be underestimated when the concentration of protein is higher than the range of the linear portion of the curve, that is, at saturation conditions. Samples that have protein concentrations higher than the concentrations in the linear range must therefore be diluted and re-assayed to obtain a more accurate estimation of the protein concentration.

The standards used most commonly for the assay are bovine serum albumin (BSA) and bovine γ-globulin (BGG). Ideally, the standard should be the same proteins in the same ratios as are found in the sample (that is, an absolute reference standard). However, for most samples this is not practical or even possible, and relative rather than absolute concentrations are sufficient in most applications.