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| Solution Information | help | |
| Enzyme: | Eukaryotic translation initiation factor 4 gamma 1 | |
| inhibitor: | BDBM48789 | |
| substrate: | n/a | |
| Solution Type: | Aqueous | |
| pH at Preparation: | n/a | |
| Temp. Prep.: | n/a | |
| Comments: | Compounds marked as active in the primary screen, AID 782, were tested in this dose-response follow-up assay. Reagents: 1. Assay buffer: 20 mM Tris buffer, pH 7.5, 50 mM NaCl, and 0.01% NP40 2. A GST-tagged fragment of eIF4GI (GST-eIF4GI (517-606)) 3. 6xHis- tagged eIF4E protein 4. Europium Chelate Anti-6xHis-Antibody: LANCE Eu-W1024 (His-Eu) from Perkin Elmer Life Sciences. 5. SureLight(tm) Allophycocyanin (APC)- anti-GST antibody (GST-APC) from Perkin Elmer Life Sciences. Procedure: 1. Make assay reaction buffer for HTS that contains recombinant GST- eIF4GI (517-606) protein (100 nM, final concentration), His-eIF4E (4.8 nM, final concentration), His-Eu (1 nM, final concentration) and GST-APC (50 nM, final concentration). 2. Dispense 4.5 uL of assay reaction buffer to 1536-well black assay plate (Costar 3724). 3. Add library compound (1 mM in DMSO) to each well to reach the appropriate final concentration and incubate plates at room temperature for 2 hr. 4. Record FRET signal | |
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