Figure 6. Dissociation parameters for inhibitor:ABL kinase binding suggest slower off-rates for ABL kinase inhibitors capable of committing cells to apoptosis following acute exposure and washout.

Purified recombinant ABL kinase protein (either unaltered or tyrosine dephosphorylated to represent the (A) active and (B) inactive conformations, respectively) was pre-incubated with a biotin-labeled antibody and Europium-labeled streptavidin in saturating concentrations of dasatinib, ponatinib, nilotinib, imatinib, DCC-2036, or staurosporine and diluted into solution containing excess Alexa-647-labeled kinase tracer. Dissociation of the inhibitor is followed by rapid binding of tracer, resulting in productive TR-FRET signal measured over time to establish dissociation curves.