The Bradford assay (sometimes referred to as the Bradford protein assay) is a common colorimetric protein assay that measures the absorbance shift of the dye Coomassie Brilliant Blue G-250 when it binds to proteins. It is used in order to quantify the total protein concentration in a sample and is widely accepted being referenced in both the USP <1057> (Biotechnology-Derived Articles – Total Protein Assay) as well as in the European Pharmacopoeia (Ph. Eur.) 2.5.33 (Total Protein).

How the Bradford Assay is Performed

The basic procedure for the Bradford assay is as follows:

1. Prepare a series of protein standards, typically using bovine serum albumin (BSA), at known concentrations
2. Add the Bradford reagent, which contains the Coomassie dye, to the protein standards and unknown samples.
3. Measure the absorbance of the samples at 595 nm using a spectrophotometer.
4. Plot a standard curve using the absorbance values of the protein standards.
5. Determine the protein concentration of the unknown samples by interpolating their absorbance values on the standard curve.

While materials for the performance of the Bradford Assay are readily available, and the test itself is reasonably straightforward, when using this assay for regulatory submissions or compliance the method must first undergo method verification / method validation to meet regulatory standards.

When to use the Bradford Assay

The Bradford assay offers numerous advantages as a protein quantification method. Its simplicity, speed, and sensitivity make it an easy choice for a number of scenarios. The method is versatile given its compatibility with various reagents and buffers, including detergents. This allows for its use across diverse applications. The assay is particularly practical due to its minimal sample volume requirements, needing only 10-100 μL.

Despite these benefits, there are certain limitations when using this test. The Bradford assay’s accuracy can vary depending on a protein’s amino acid composition, as the dye-protein binding interaction isn’t uniform across different proteins. This variability is pronounced for proteins rich in basic or aromatic amino acids, which tend to bind more strongly with the dye. Additionally, certain reducing agents and other substances can interfere with the assay, potentially compromising its accuracy, and necessitating the use of alternative approaches

For clients looking for a Bradford assay testing lab, Nucro-Technics is an excellent choice. The test is run routinely and is most commonly validated and utilized in GMP product release scenarios. It has also also found use for the characterization of test articles in order to support non-clinical GLP scopes of work.