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The protein biotinylating agent, pentafluorophenyl 6-(biotinylam ido)hexanoate (3) was synthesized. A straightforward synthetic route, biotin, biotin pentafluorophenyl ester (1), 6-(biotinylam ido)hexanoate (2) and pentafluorophenyl 6-(biotinylam ido)hexanoate (3) was followed. This agent was used for biotin-labelling of different proteins, bovine serum album in, immunoglobulin, horseradish peroxidase and β-galactosidase. Optimal conditions for the labelling were determined, as well as the influence of biotin-tagging to the biological activity of the proteins. An optimal ratio of the biotinylating agent to protein resulted in effective biotinylation without a significant decrease in the activity of the labelled enzyme. The labelled proteins proved to be suitable for immunodetection of proteins in both microplate and Western blot immunohistochemistry, as well as in a competitive steroid ELISA of testosterone and 20-hydroxyecdysone.

Synthesis and Use of Pentafluorophenyl 6-(Biotinylamido)hexanoate An Alternative Reagent for Labelling of Proteins with Biotin Moiety