RESUMO
Coomassie brilliant blue R-250 (CBB) is a popular and widely used dye for detection of proteins by gel electrophoresis. However, commercially available CBBs are complex mixtures of numerous chromogenic compounds that vary from lot to lot, thereby giving an undesirable level of variation in reproducibility, precision, and specificity in staining gels. We have developed a silica gel column chromatographic method for purification of commercial CBBs in high yield and have standardized each lot to perform equivalently in staining proteins as determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and quantitative scanning densitometry. This is a major improvement in protein purity determinations by quantitative scanning densitometry. A thinlayer chromatographic method for quality control testing of the purified CBB lots was also developed. Plasma desorption mass spectrometry was used to identify components of silica gel column fractions. Scanning densitometry was the technology used to establish performance equivalency between different CBB preparations. The less polar chromogenic compounds are nonblue and/or fluorescent in color, contain mono- or unsulfonated structures, and lack significant protein binding capacity. The more polar chromogenic compounds are green and blue-green in color, contain tri- and tetrasulfonated moieties, compared to the disulfonated structure of CBB, and bind to protein at least 40 times more effectively than pure CBB. The concentrations of these highly polar chromogens differ from lot to lot and act as "inhibitors" in protein staining, thereby causing variability in protein staining.
