| Solution Information | help | |
| Enzyme: | Ephrin type-A receptor 4 | |
| inhibitor: | BDBM33087 | |
| substrate: | n/a | |
| Solution Type: | Aqueous | |
| pH at Preparation: | n/a | |
| Temp. Prep.: | n/a | |
| Comments: | Materials: 1)Assay buffer (x2) contained 2M diethanolamine, pH 9.8, 1 mM MgCl2. 2)Secreted alkaline phosphatase (SEAP) working solution contained SEAP-EphA4 fusion construct, also utilized in EphA4 screen (AID 689). SEAP was diluted in the assay buffer (x2) at 1/100 dilution. 3)pNPP working solution contained 2.27 mg/ml pNPP (Pierce) in mQ water. Protocol: 1)2.5 uL of compounds or control solutions added to 384-well plate: a)controls (10% DMSO) to columns 1-2 and 23-24 b)serially diluted compounds to columns 3-22 2)11 uL assay buffer and SEAP working solution were added to columns 1-2 and 3-24, respectively. 3)11 uL pNPP working solution was added to the whole plate after 15 min of preincubation at room temperature. 4)Reaction was stopped after incubation for 1h at room temperature with 25 uL 1M NaOH. 5)Absorbance at 405 nm was measured on an EnVision plate reader (Perkin Elmer). 6)Data analysis was performed using sigmoidal dose-response equation through non-linear regres | |
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