| Solution Information | help | |
| Enzyme: | Eukaryotic translation initiation factor 4H | |
| inhibitor: | BDBM40537 | |
| substrate: | n/a | |
| Solution Type: | Aqueous | |
| pH at Preparation: | n/a | |
| Temp. Prep.: | n/a | |
| Comments: | HTS Assay Protocol Assay materials: 1) His6-eIF4H (Obtained from Assay Provider) 2) 5'-fluorescein-labeled poly(U)10 (Purchased from Dharmacon) 3) Assay Buffer: 5 mM Hepes pH 7.5, 0.005 % Tween-20 Procedure: 1. Dispense 2 ul of assay buffer into columns 1 and 2 of a black, Corning (#2725) 1536 well assay plate. 2. Add 2 ul of eIF4H protein, 30 nM final concentration into columns 3-48. 3. Add 2 ul of 5'-fluorescein-labeled poly(A)10 2.5 nM final concentration into columns 1-48. 4. Using a HighRes biosolutions pintool dispense 70 nl of 2 mM compounds in DMSO to columns 5-48. 5. Using a HighRes biosolutions pintool dispense 70 nl of DMSO to columns 1-4. 6. Read plate on a BMG PHERAstar at 485/520/520nm in Fluorescence Polarization mode. i. Positioning delay = 0.0 ii. Flashes/well = 10 7. Data analysis was performed using CBIS software (ChemInnovations, Inc). 8. Fluorescence intensity of each sample was normalized to the average fluorescence intensity value of the plate negativ | |
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