Optimization of erythrocyte membrane glycoprotein fluorescent labeling with dansylhydrazine after polyacrylamide gel electrophoresis.
An improved procedure for the labeling of glycoproteins with dansylhydrazine subsequent to electrophoresis in polyacrylamide gels is reported. This procedure is derived from the work of Eckhardt et al. (1976, Anal. Biochem. 73, 192-197) and Weber and Hof (1975, Biochem. Biophys. Res. Commun. 65, 1298-1302) who showed that dansylhydrazine may be condensed with the aldehyde groups of oxidized glycoprotein carbohydrates and the resulting hydrazones reduced with dimethylamine borane and/or sodium borohydride. Using the known distribution of erythrocyte membrane glycoproteins as a benchmark the effect of variation of a number of process parameters was investigated and an optimal procedure identified. The procedure is shown to be relatively insensitive to moderate variations in reagent composition, pH, and time of incubation with dansylhydrazine solution or reducing agents. It is also shown that labeling patterns may be preserved in dried gels if dimethylsulfoxide is replaced or omitted from all of the process solutions and destaining is effected with 1 M sodium acetate, pH 5.6. While specifically developed for the labeling of erythrocyte membrane proteins, the procedure is demonstrated to be applicable to other glycoprotein containing preparations.
D003619:Dansyl Compounds; D004121:Dimethyl Sulfoxide; D004591:Electrophoresis, Polyacrylamide Gel; D004910:Erythrocyte Membrane / Q000032:analysis*; D006023:Glycoproteins / Q000097:blood*; D006801:Humans; D006834:Hydrazines; D006863:Hydrogen-Ion Concentration; D013050:Spectrometry, Fluorescence
D003619:Dansyl Compounds; D006023:Glycoproteins; D006834:Hydrazines; C027913:dansyl hydrazine; D004121:Dimethyl Sulfoxide