| Solution Information | help | |
| Enzyme: | POsterior Segregation | |
| inhibitor: | BDBM52882 | |
| substrate: | n/a | |
| Solution Type: | Aqueous | |
| pH at Preparation: | n/a | |
| Temp. Prep.: | n/a | |
| Comments: | 2024-02-W01-04 7.5 nL of compound per well was pre-dispensed into black Aurora 1536 low-base plates (PN 00029844) in duplicate assay plates. Predispensed plates were stored lidded at room temperature for up to two weeks. For higher doses, 15 or 30 nL was predispensed. POS-1 protein, expressed in E. coli JM109 with a C-terminal fusion to maltose binding protein, was prepared by the assay provider at approximately 50 uM as previously described. A protein master mix was prepared at 200 nM in 1x buffer (20 mM tris pH 8.0 (Ambion), 40 mM NaCl (Ambion), 0.004% IPEGAL (Sigma), 100 uM zinc acetate dihydrate (Fluka)). The solution was loaded on the dispenser (Combi nL, Thermo) at room temperature. The target binding sequence is a 3'-UTR fragment, mex3. Fluorescent mex3 RNA (5'-AACUAUUAUUAUUUGUUAUUCAUAUUUU-FITC-3', IDT) mastermix was prepared in 1x buffer and supplemented with 0.02 mg/mL tRNA type X (Sigma). The RNA mastermix was heated at 65 degrees for 2 minutes then coole | |
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