Quantitative electrophoretic analysis of proteins labeled with monobromobimane

Protein cysteine residues were labeled with the thiol-specific fluorescent probe monobromobimane prior to electrophoresis. Labeling was rapid and complete under the conditions employed. Scanning the negative films of photographed gels with a laser densitometer provided quantitation. Analysis of five different cysteine-containing proteins demonstrated that the fluorescence was dependent on the total number of cysteine residues present for each protein and independent of the protein species. The method allowed the construction of two nomograms for the universal quantitative analysis of cysteine-containing proteins. One nomogram calculated the limit of detection for each protein based on the protein's molecular mass, the protein's molar ratio of cysteine, and a nominal detection limit of 10 pmol cysteine. The second nomogram determined the quantity of the class 4 protein in outer membrane extracts of Neisseria meningitidis from the protein's molecular mass, its molar ratio of cysteine, and its total cysteine content. Additional applications for the analysis of protein cysteine and cystine content are discussed.