The Bradford assay is a protein determination method that involves the binding of Coomassie Brilliant Blue G-250 dye to proteins . The dye exists in three forms: anionic - blue, cationic - red, and neutral - green.
In the acidic environment of the reagent, Under acidic conditions, the dye is predominantly in the doubly protonated red cationic form. When protein binds to the Coomassie dye, it results in a spectral shift from the reddish/brown form of the dye to a stable un-protonated blue form. The blue dye form is measured using a spectrophotometer or microplate reader
Development of color in Bradford protein assays is associated with the presence of certain basic amino acids (primarily arginine, lysine and histidine) in the protein. The principal for protein binding between dye and Protein is described by Van der Waals forces and hydrophobic interactions. The number of Coomassie dye bound to each protein is approximately proportional to the number of positive charges found on the protein.
Free amino acids, peptides and low molecular weight proteins do not produce color with Coomassie dye reagents. Generally, the mass of a peptide or protein must be at least 3000 daltons to be detectable with this reagent.
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