What Is the Principle Behind Protein Quantification Using the Coomassie Brilliant Blue Assay?
The Coomassie Brilliant Blue (CBB) staining method is a widely used technique for determining protein concentration. Its underlying principle is based on the reversible binding interaction between Coomassie Brilliant Blue dye and proteins. Upon binding to proteins, the dye undergoes a shift in its absorbance characteristics, with its maximum absorbance wavelength changing from 465 nm to 595 nm.
Under acidic conditions, partial protonation of the phosphate groups in the dye imparts a positive charge, while certain amino acid residues in proteins—particularly arginine and lysine—carry negative charges. This results in electrostatic interactions that facilitate the binding of the dye to the protein molecules.
By constructing a standard curve using known concentrations of a reference protein, the absorbance of unknown samples can be measured at 595 nm using a spectrophotometer. The protein concentration in the sample is then estimated by comparing the measured absorbance to the standard curve.
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