Mechanism of Coomassie brilliant blue G-250 binding to proteins: a hydrophobic assay for nanogram quantities of proteins
We investigated the mechanism of Coomassie brilliant blue G-250 (CBB) binding to proteins in order to develop a protein assay with the maximum possible sensitivity. We found that the neutral ionic species of CBB binds to proteins by a combination of hydrophobic interactions and heteropolar bonding with basic amino acids. On the basis of these findings, we developed a very sensitive hydrophobic assay for proteins (at the nanogram level) using the hydrophobic reagents ammonium sulfate and trichloroacetic acid under pH conditions that increase neutral species concentration in the assay reagent in order to enhance the binding of more CBB dye molecules per protein molecule than in previous CBB-based assays.
KeywordsCoomassie brilliant blue G-250 Proteins Quantification
- 2.Lowry OH, Rosebrough NJ, Farr AL, Randall RJ (1951) J Biol Chem 193:265–275Google Scholar
- 11.Sigma-Aldrich (2004) Technical bulletin of Sigma Bradford assay (product no B6916). http://www.sigmaaldrich.com/sigma/bulletin/b6916bul.pdf. Accessed 9 Dec 2007
- 12.Sigma-Aldrich (2006) Technical bulletin of Sigma BCA assay (product no B9643). http://www.sigmaaldrich.com/sigma/bulletin/b9643bul.pdf. Accessed 9 Dec 2007
- 13.Pierce (2006) Pierce protein assay technical handbook. http://www.piercenet.com/files/1601325%20ProteinAssay.pdf. Accessed 9 Dec 2007