| Abstract: | A biotin-containing, thiol-specific reagent, 3-(N-maleimido-propionyl) biocytin (MPB), was synthesized and used to biotinylate various proteins via native or artificially induced sulfhydryl groups. In combination with appropriate avidin- or streptavidin-conjugated markers (i.e., fluorescent, enzyme-conjugated, electron-dense, etc.), MPB essentially constitutes a universal, multipurpose, thiol-specific probe. The reagent could be used to detect protein SH groups on dot blots with sensitivities in the femtomole range. The labeling was very specific for sulfhydryl groups or reduced S-S bonds; proteins lacking free SH groups were unlabeled by this method. Due to the long spacer between the biotinyl group and the reactive maleimide, improved adsorption of biotinylated proteins to avidin columns was achieved. An SH-containing enzyme (beta-galactosidase) was biotinylated with MPB, and the resultant biotinylated enzyme could be used as an efficient histochemical probe. The use of this reagent is recommended to biotinylate proteins which contain nonessential SH groups or which can be easily thiolylated prior to reaction with MPB. |
