Assay Method Information

Assay Name:  HPK-1 biochemical enzyme assay
Description:  Compound inhibitory potency was measured in an HPK-1 kinase inhibition assay. Briefly, recombinant full length HPK-1 enzyme (6.8 nM) was incubated with 10 μM ATP and 12.5 μM swine myelin basic protein (MBP) for 30 mins. at 25° C. in the presence of various concentrations of test compound or vehicle in 40 mM Tris.Cl pH7.4 buffer containing 20 mM MgCl2, 50 μM DTT and 0.1 mg/mL BSA. Reactions were quenched and reaction mixtures were then analyzed by an ADP-Glo kit which measures the formed ADP. The percent inhibition was calculated from the substrate conversion considering no enzyme control reactions for 100% inhibition and vehicle only reactions for 0% inhibition. The compounds were dissolved in DMSO and evaluated at 10 concentrations to determine an IC50 value.
Affinity data for this assay
 

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