Table 1Final 1536-well assay protocol

StepParameterValueDescription
1Reagent3 μLPEP/hPK-M2 buffer (0.5 mM and 0.1 nM final, respectively)
2Controls23 nlActivator control (NCGC00031955)
3Library compounds23 nl57 μM to 0.4 nM dilution series
4Reagent1 μLADP buffer (0.1 mM final)
5Incubation time1 hrRoom temperature
6Reagent2 μLKinase-Glo
7Assay readoutluminescenceViewLux
Step Notes
1Black walled clear bottom Greiner white solid plates; 4 tips dispense to all wells except column 3 of buffer 0.133 nM hPK-M2, 0.67 mM PEP, 50 mM Imidazole pH 7.2, 50 mM KCl, 7 mM MgCl2, 0.01% Tween, 0.05% BSA. Column 3, 1 tip of same buffer without hPK-M2.
2Column 1, NCGC00031955 Activator titration 57 μM start, 16 points in duplicate 1:2 dilutions; Column 2 neutral, DMSO only; Column 3 no enzyme; Column 4, top 16 rows are NCGC0031955 at 57 μM, bottom 16 rows are DMSO only.
3Pintool transfer (tip wash sequence; DMSO, iPA, MeOH, 3-s vacuum dry)
4ADP in same 4 tips dispense to all wells of buffer containing 0.4 mM ADP (final assay 0.1 mM), 50 mM Imidazole pH 7.2, 50 mM KCl, 7 mM MgCl2, 0.01% Tween, 0.05% BSA.
5Plates covered with stainless steel rubber gasket-lined lids containing pin holes for gas exchange
6Kinase-Glo detection. Luciferase-based detection of ATP product

From: Identification of activators for the M2 isoform of human pyruvate kinase

Cover of Probe Reports from the NIH Molecular Libraries Program
Probe Reports from the NIH Molecular Libraries Program [Internet].

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