Figure 7. Stoichiometry of site-specific biotin incorporation.

Figure 7

Stoichiometry of site-specific biotin incorporation. Three derivatives of the E. coli EF-Tu gene containing His-Tag encoding sequences at the 3'-end and amber codons at positions 2 (amb2), 184 (amb184) and 396 (amb396), respectively, were used as templates for cell-free protein biosynthesis with the EasyXpress Site-Specific Biotin Kit. Following protein synthesis aliquots from the reactions were incubated with and without streptavidin and subjected to native polyacrylamide gel electrophoresis. Subsequently proteins were analyzed by western blotting using an antibody against His-Tag (Penta His HRP conjugate). Irrespective of the amber codon position synthesized EF-Tu was almost completely shifted by streptavidin indicating a stoichiometric biotin incorporation (i.e., each protein molecule contains one biotin moiety).

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

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