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. Author manuscript; available in PMC: 2019 Oct 15.
Published in final edited form as: Bioorg Med Chem. 2018 Apr 4;26(19):5280–5290. doi: 10.1016/j.bmc.2018.04.009

Figure 2. Labeling QC-sfGFP-FLAG via the quadricyclane ligation.

Figure 2

A) Schematic showing labeling QC-sfGFP-FLAG with 8. B) Structure of QC acetate (QCOAc, 9). C) Purified QC-sfGFP-FLAG or purified Boc-sfGFP-FLAG was incubated with 200 μM 8 in PBS with 1 mM K3Fe(CN)6 (+8) or with 1 mM K3Fe(CN)6 in PBS (−8) for 1 h at rt in the dark. The reaction was quenched with QCOAc (100 equiv. relative to 8) followed by treatment with sodium diethyldithiocarbamate (Na-DEDTC). Protein was analyzed by western blot with an α-biotin-HRP antibody. D) Various quantities of purified QC-sfGFP-FLAG or Boc-sfGFP-FLAG (5.0, 2.5, 1.0, 0.5, or 0 μg) were added to 10.0 μg bacterial lysate. The mixtures were incubated with 8 (200 μM) in K3Fe(CN)6 (1 mM) in PBS for 1 h at rt, quenched via addition of QCOAc (100 equiv. relative to 8), and treated with Na-DEDTC. Mixtures were analyzed via western blot with an α-biotin-HRP antibody.