The Bradford protein assay is an easy and simple method for protein quantification of your protein concentration, yet may still require troubleshooting occasionally.
The Coomassie Brilliant Blue dye binds to both basic and aromatic amino acid residues, resulting in a change in color from brown to blue, and an absorbance shift. The concentration of your protein can be determined by referencing a standard protein, most commonly BSA (Bovine serum albumin).
Factors such as: temperature, wavelength, detergents and even the type of cuvettes you use can influence the measurement and provide incorrect results. Below we offer some useful tips to avoid these issues as well as solutions for when they arise.
Problem: Absorbance of the protein sample is lower than expected
One of the major problems encountered when performing a Bradford assay is the presence of interfering substances in the buffer. Table 1 summarizes a list of the most commonly used substances and their compatible concentrations. For detailed information, please refer to the manufacturers’ protocol.
Table 1: Compatible substance concentrations in Bradford assay (modified after [1] Quick Start Bradford Protein Assay – Instruction Manual, #4110065, Bio-Rad; [2] Protein assay compatibility table, Tech tip #68, (2012))
Note: This is not a comprehensive list of compatible substances and their concentrations. There are many substances that can affect proteins in different ways. Reagents may also change the pH of the assay and interfere with the Bradford assay.