Coomassie Blue dye

The Bradford protein assay as described in Chapter 2 is based on the absorbance change that occurs upon binding of Coomassie Blue dye to proteins. Explain how you would study the dynamics of this binding process and experimentally determine the number of binding sites on a protein. 

Neuhoff, V., Stamm, R., and Eibl, H. (1985) Clear background and highly sensitive protein staining with Coomassie Blue dyes in polyacrylamide gels a systematic analysis. Electrophoresis 6, 427-448. 

Another assay, the Bradford assay, also known as the Coomassie dye binding method, was first described in 1976 [19]. In an acidic environment, proteins will bind to Coomassie dye and cause a shift from the reddish brown color (465 nm) to the blue dye protein complex read at 595 nm. The development of the color is attributed to the presence of the basic amino acids arginine, lysine, and histidine. Van der Waals forces and hydrophobic interactions account for the dye binding and the number of Coomassie blue dye molecules bound is roughly proportional to the number of positive charges on the molecule. A protein molecular weight of about 3 kDA is required for successful color development. The Bradford assay dose response is nonlinear and this method demonstrates the greatest difference in reactivity with BSA compared to BGG. 

After IEF electrophoresis, ampholytes have to be removed from gels before staining can be carried out, because the staining agents commonly used are bound by the ampholytes. Removal of the ampholytes is effected by soaking gels hi 5% trichloroacetic acid, and staining is carried out with Coomassie Blue dye. 

To estimate the release rate of insulin in vitro, a pellet made of 20% insulin in palmitic acid was broken and a quarter piece was dropped into a 250 mL Erlymer flask Containing 100 mL PBS. In another run, 250 mL of a 0.1 M orthophosphate solution at pH 2.4 was used with an 1/8-size piece. At daily or weekly intervals, depending on the rate of release, an aliquot of 0.8 mL of the stirred solution was withdrawn and mixed with 0.2 mL of Coomassie blue dye solution for protein analysis. After 30 min at room temperature, the intensity of the blue solution was read at 660 nm. The sensitivity of this method is 1 pg protein per mL. Since the solution was kept at 21 C, bacterial fouling would affect the dissolved protein. Changing the solution every 5 days was found to correct this problem, and the amount of protein entered the fresh solution was added to the total as the study continued. 

Coomassie blue dye binding assay This protein-determination method involves the binding of... 

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