Skip to main content
. Author manuscript; available in PMC: 2019 Dec 21.
Published in final edited form as: ACS Chem Biol. 2019 Nov 14;14(12):2793–2799. doi: 10.1021/acschembio.9b00678

Figure 5.

Figure 5.

Tetrazine-mediated activation of isocyano-caged lipoic acid ligase-acceptor peptide (LAP) tag in living cells. (A) The reaction scheme for tetrazine-mediated activation of isocyano-caged proteins. NCK was site-specifically incorporated into the LAP-tagged neurexin1β (NRX-LAP) to block the critical lysine residue. The NCK-modified LAP tag was then decaged by tetrazine treatment, followed by reaction with coumarin fluorophore catalyzed by lipoic acid ligase (LplA). (B) HEK293T cells expressing NRX-LAP or NRXLAP-TAG with or without NCK were mixed with 1 mM dpTz in PBS for 20 min, followed by the incubation with 1 mM pyrrolidine for 8 h. After PRIME labeling (blue) and cell fixation, 0.1 μg/mL anti-Myc antibody and 7.5 μg/mL FITC-conjugated secondary antibody (green) were added to check the expression level of NRX-LAP. Nucleus staining was carried out with 5 μM DRAQ5 (red) before confocal imaging. Scale bars = 25 μm.