PURIFICATION OF COMMERCIAL COOMASSIE BRILLIANT BLUE R-250 AND CHARACTERIZATION OF THE CHROMOGENIC FRACTIONS

Coomassie brilliant blue R-250 (CBB) is a popular and widely used dye for detection of proteins by gel electrophoresis, However, commerciall y available CBBs are complex mixtures of numerous chromogenic compound s that vary from lot to lot, thereby giving an undesirable level of va riation in reproducibility, precision, and specificity in staining gel s, We have developed a silica gel column chromatographic method for pu rification of commercial CBBs in high yield and have standardized each lot to perform equivalently in staining proteins as determined by sod ium dodecyl sulfate-polyacrylamide gel electrophoresis and quantitativ e scanning densitometry. This is a major improvement in protein purity determinations by quantitative scanning densitometry. A thin-layer ch romatographic method for quality control testing of the purified CBB l ots was also developed. Plasma desorption mass spectrometry was used t o identify components of silica gel column fractions. Scanning densito metry was the technology used to establish performance equivalency bet ween 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 p olar chromogenic compounds are green and blue-green in color, contain tri- and tetrasulfonated moieties, compared to the disulfonated struct ure of CBB, and bind to protein at least 40 times more effectively tha n pure CBB. The concentrations of these highly polar chromogens differ from lot to lot and act as ''inhibitors'' in protein staining, thereb y causing variability in protein staining. (C) 1996 Academic Press, In c.