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. Author manuscript; available in PMC: 2013 Sep 1.
Published in final edited form as: Nat Chem. 2012 Feb 5;4(4):298–304. doi: 10.1038/nchem.1250

Figure 3. Characterization of tetrazine-norbornene reactions.

Figure 3

a, `Turn-on' fluorescence of tetrazine fluorophores 9 (i) and 13 (ii) on reaction with 5-norbornene-2-ol (Nor). b, Specific and quantitative labelling of sfGFP that bears 2, demonstrated by SDS–PAGE (Coomassie staining and in-gel fluorescence) (i) and mass spectrometry (ii) before bioconjugation (red spectrum, found 27,975.5±1.5 Da, expected 27,977.5 Da) and after bioconjugation (blue spectrum, found 28,783.0±1.5 Da, expected 28,784.4 Da). c, Specificity of labelling 2 in sfGFP versus the E. coli proteome. Lanes 1–5: Coomassie-stained gel showing proteins from E. coli producing sfGFP in the presence of the indicated concentration of unnatural amino acids 2 or 3. Lanes 6–10: The expressed protein was detected in lysates using an anti-His6 antibody. Lanes 11–20: fluorescence images of protein labelled with the indicated fluorophore 12 or 13. d, Labelling of myoglobin that bears 2 at position 4 with fluorophore 12: fluorescence imaging (top) and Coomassie-stained loading control (bottom).