| Solution Information | help | |
| Enzyme: | 14-3-3 protein gamma | |
| inhibitor: | BDBM39874 | |
| substrate: | n/a | |
| Solution Type: | Aqueous | |
| pH at Preparation: | n/a | |
| Temp. Prep.: | n/a | |
| Comments: | Compounds identified as active from the primary screen, AID 422 - HTS for 14-3-3 protein interaction modulators, were selected for testing in this assay. Each compound was tested at 6 concentrations, 1:2 serial dilutions starting at a nominal test concentration of 50 micromolar. Each concentration was tested in triplicate. In some cases, compounds were retested in 6 concentration, 1:2 serial dilutions starting at 25 micromolar or lower, to obtain the final IC50. These cases are noted in the PUBCHEM_ASSAYDATA_COMMENT field. The recombinant hexaHis-tagged 14-3-3gamma were expressed and purified as previously described (Fu et al, PNAS, 1993). The peptide sequence containing phosphorylated S259 of Raf-1 was synthesized and labeled with 5/6 carboxytetramethylrhodamine. HEPES buffer (10mM, pH 7.4), NaCl (150mM), Tween-20 (0.05%), and DTT (0.5 mM) was used through the screening. The 14-3-3 FP assay was carried out in black 384-well uplates in a total volume of 50uL in each well. 49uL | |
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