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.
Georgiou
Christos D
CD
Department of Biology, Section of Genetics, Cell Biology and Development, University of Patras, 26100, Patras, Greece. c.georgiou@upatras.gr
Grintzalis
Konstantinos
K
Zervoudakis
George
G
Papapostolou
Ioannis
I
D000645:Ammonium Sulfate / Q000737:chemistry; D000818:Animals; D006863:Hydrogen-Ion Concentration; D057927:Hydrophobic and Hydrophilic Interactions; D011485:Protein Binding; D011506:Proteins / Q000032:analysis* / Q000737:chemistry*; D012394:Rosaniline Dyes / Q000737:chemistry*; D012680:Sensitivity and Specificity; D014238:Trichloroacetic Acid / Q000737:chemistry
D011506:Proteins; D012394:Rosaniline Dyes; D014238:Trichloroacetic Acid; C004692:coomassie Brilliant Blue; D000645:Ammonium Sulfate