| Solution Information | help | |
| Enzyme: | Polyadenylate-binding protein 1 | |
| inhibitor: | BDBM73184 | |
| substrate: | n/a | |
| Solution Type: | Aqueous | |
| pH at Preparation: | n/a | |
| Temp. Prep.: | n/a | |
| Comments: | HTS Assay Assay materials: 1) GST-PABP RRM1/2 (Obtained from Assay Provider) 2) 5'-fluorescein-labeled poly(A)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 PABP protein, 15 nM final concentration into columns 3-48 3. Add 2 ul of 5'-fluorescein-labeled poly(A)10 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 negative control w | |
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