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. Author manuscript; available in PMC: 2020 Jul 31.
Published in final edited form as: J Am Chem Soc. 2019 Jul 5;141(30):11912–11922. doi: 10.1021/jacs.9b02963

Figure 2.

Figure 2.

Rapid and mild enrichment of protein kinases with TCO-conjugated probes. (A) Schematic for conversion of 1 (left) into 1-TCO (right). (B) Co-crystal structure of 1 bound to Src V284C (PDB ID: 5SWH). (C) Inhibition curves and Ki values of 1 and 1-TCO against recombinant Src V284C activity. Values shown are mean ± SEM, n = 3. (D) Schematic representation of kinase enrichment with TCO-conjugated probes. (E) 1-TCO selectively enriches a drug-sensitized Src mutant (Src V284C) from cell lysates. HEK293 cell lysates containing either Src V284C or Src WT were treated with 1-TCO (5 μM), incubated with tetrazine-conjugated sepharose beads (Tz-beads), and captured proteins were eluted under reducing and denaturing conditions. A Src immunoblot of the flow through (FT) and elution (EL) are shown. (F) 1-TCO selectively enriches Src V284C from cells. HEK293 cells expressing either Src V284C or Src WT were treated with the indicated concentrations of 1-TCO, lysed, and then incubated with Tz-beads. Captured proteins were eluted under reducing and denaturing conditions. A Src immunoblot of the flow through (FT) and elution (EL) are shown.