| Assay Method Information | |
| | The p38alpha:eIF4E-BP1 biochemical assay |
| Description: | In a 10 μL reaction, the compounds of various concentrations (10 nL) were pre-incubated with 3 nM active p38 at (Abcam #ab271606 or in-house prepared: Avi-Tev-8HIS-p38a phosphorylated by GST-MKK6) for 15 minutes at room temperature following by the addition of 120 nM ULight-eIF4E-binding protein 1 (Thr37/46) peptide (Perkin Elmer #TRF-0128M) and 100 M ATP (Fisher Scientific #B320). After 1 h incubation at room temperature, 5 μL detection reagent (50 mM EDTA, TX Lance reaction buffer (Perkin Elmer #TRFLAB100) and 2 nM Lance Ultra Europium-anti-phospho-eIF4E-binding protein 1 Thr37/46 (Perkin Elmer #TRF-0216M)) were added to the reaction and incubated for 1 h at room temperature. HTRF signal (the ratio between emission at 620 nm and emission at 665 nm upon excitation at 330 nm) was read by TECAN Spark microplate reader. The signal was normalized to vehicle control (DMSO, 0% inhibition) and 1 μM GS-703447 (100% inhibition) to generate IC50 inhibition as a function of compound concentration and filled in a four-parameter logistic equation to generate IC50. |
| Affinity data for this assay | |
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