Figure 10. Site-specific incorporation of a fluorescent moiety.

Figure 10

Site-specific incorporation of a fluorescent moiety. Genes for C-terminally His-tagged EF-Ts, N-terminally His-tagged tandem ubiquitin and FABP containing amber codons at positions 47, 2 and 88, respectively, were expressed in the EasyXpress Site-Specific Biotin Kit (QIAGEN GmbH, Hilden, Germany). Instead of biotinylated amber suppressor tRNA supplied with the Kit an amber suppressor tRNA that we charged chemically with the fluorophor Bodipy-TMR (Invitrogen GmbH, Karlsruhe, Germany) was added to the reactions. All reactions were performed in the presence of radiolabeled leucine (14C). The synthesized proteins were separated by SDS-PAGE and radioactive as well as fluorescent bands visualized by using a Typhoon 8600 variable mode imager. As can be seen from the fluorogram (right panel) BODIPY-TMR has been incorporated into all model proteins. Termination products (indicated by asterisks) are only visible in the autoradiogram, since they contain no fluorescent moiety.

From: Cell-Free Synthesis of Defined Protein Conjugates by Site-directed Cotranslational Labeling

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