| Assay Method Information | |
| | Biochemical Assay for GCN2 |
| Description: | Activity of GCN2 kinase was determined using a TR-FRET kinase activity assay (e.g. Riddle et al. Analytical Biochemistry (2006) 356(1) 108-116). Assays were conducted in 384-well plates (13 μL assay volume) using 2 nM GCN2 (Carna Biosciences), 130 nM GFP-EIf2α (Invitrogen), 0.2 mg/mL E. coli tRNA (sigma) and 1 mM ATP in kinase buffer (Invitrogen). Inhibition of GCN2 was measured by adding serial diluted test compound (final assay concentration of 0.5% DMSO) followed by a 3-hour incubation. Tb-peIF2α (pSer52) antibody (Invitrogen) (2 nM final assay concentration) in kinase buffer containing EDTA (final assay concentration of 20 mM) was added. After a 60 min incubation at room temperature, TR-FRET was monitored using an excitation wavelength of 340 nm and emission wavelengths of 490 nm and 520 nm. The emission ratio (520/490) at each compound concentration of was converted to percent inhibition using controls (i.e., reaction with no test compound and reaction with a known inhibitor) and IC50 values were calculated by fitting a four-parameter sigmoidal curve to the data using Prism (GraphPad software). |
| Affinity data for this assay | |
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