User:Vanessa Arcos Borja/Bradford protein assay

The Bradford protein assay (also known as the Coomassie protein assay) was developed by Marion M. Bradford in 1976. It is a quick and accurate spectroscopic analytical procedure used to measure the concentration of protein in a solution. The reaction is dependent on the amino acid composition of the measured proteins. The main goal of this assay is to detect the binding of protein molecules to Coomassie dye under acidic conditions, which results in a color change from brown to blue. This method detects the presence of basic amino acid residues, namely arginine, lysine, and histidine, which facilitate the formation of the protein-dye complex.To ensure the assay is properly functioning, it must be conducted at an acidic pH level (2). This acidic environment is necessary for the dye to protonate and absorb light at 465 nm in solution.

Unlike other protein assays, the Bradford protein assay is less susceptible to interference by various chemical compounds, such as sodium, potassium, or even carbohydrates like sucrose, that may be present in protein samples. As a result, new modifications for an improved Bradford Protein Assay have been underway that specifically focus on enhancing detection accuracy for collagen proteins. One notable modification involves incorporating small amounts, approximately 0035%, of sodium dodecyl sulphate (SDS). An exception of note is elevated concentrations of detergent. Sodium dodecyl sulfate (SDS), a common detergent, may be found in protein extracts because it is used to lyse cells by disrupting the membrane lipid bilayer and to denature proteins for SDS-PAGE. While other detergents interfere with the assay at high concentration, the interference caused by SDS is of two different modes, and each occurs at a different concentration. When SDS concentrations are below critical micelle concentration (known as CMC, 0.00333%W/V to 0.0667%) in a Coomassie dye solution, the detergent tends to bind strongly with the protein, inhibiting the protein binding sites for the dye reagent. This can cause underestimations of protein concentration in solution. When SDS concentrations are above CMC, the detergent associates strongly with the green form of the Coomassie dye, causing the equilibrium to shift, thereby producing more of the blue form. This causes an increase in the absorbance at 595 nm independent of protein presence.