Why is it necessary to have the dye in excess when you perfo

Why is it necessary to have the dye in excess when you perform the Bradford Assay?

Solution

Bradford assay is a quantitative assay used to calculate the concentration of protein in a sample. The dye used in Bradford assay is coomassie blue.

This dye is used in excess because it exists in two-forms: cationic unstable and anionic stable. The cationic unstable dye is reddish-brown in appearance, firstly it binds with the protein in the sample and forms and unstable protein-dye mixture. Afterwards, the unstable protein complex binds with stable dye which is blue in color and forms stable protein. The protein solution will appear blue. More the amount of protein, more will be the blue color of the dye-protein complex. So, the dye is consumed twice.