| Assay Method Information | |
| | Biochemical Assay for Modulation (Inhibition/Activation) of GCN2 |
| Description: | GCN2 protein was obtained from Carna Biosciences (cat #05-153). The protein was diluted in assay buffer (ThermoFisher Scientific, #PV6135), 2 mM dithiothreitol (DTT), to obtain a final concentration of 2 nM and 5 μL was plated in a 384-well white assay plate. Test compounds were serially diluted to 11 concentrations by 3-fold dilution in DMSO and 10 nL of stock was plated into 384 well white assay plate. DMSO was used as a vehicle control. GFP-eIF2a protein was obtained from ThermoFisher (cat #PV4809). The protein was diluted in assay buffer to a 2× concentration of 200 nM along with 300 μM ATP (final concentration of 100 nM GFP-eIF2a and 150 μM ATP) in the presence of 2 mM DTT and a 5 μL aliquot was added to each well containing the GCN2 protein and test compound. The plate was incubated in the dark at 25° C. for 1.5 hours, shaking at 1250 rpm. Tb-anti P-eIF2a (ThermoFisher cat #PV4815) was diluted to a to a concentration of 1 nM in TR-FRET Dilution Buffer (ThermoFisher cat #PV3574). 10 μL of the Tb-anti P-eIF2a solution was added to the TR-FRET reaction. The plate was incubated in the dark for 2 h at 25° C. shaking at 600 rpm. |
| Affinity data for this assay | |
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