| Solution Information | help | |
| Enzyme: | Ubiquitin carboxyl-terminal hydrolase BAP1 | |
| inhibitor: | BDBM40267 | |
| 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 436 - HTS for BAP1 Enzyme Inhibitors, were selected for testing in this assay. Each compound was tested in 6 concentration, 1:2 serial dilutions starting at a nominal test concentration of 50 micromolar. Each concentration was tested in triplicate. Procedure: 1. Dilute BAP1 and Ub-AMC (provided by investigator) using Tris-HCl Buffer (50 mM Tris-HCl, 100 ug/ml ovalbumin, pH 7.4, 10 mM DTT). 2. Add 25 ul of BAP1 (5 nM) to 384-well black plate with appropriate controls. 3. Transfer compounds (2 mM in DMSO) to 384-well plate to arrive at final compound concentrations. 4. Add 25 ul of 800 nM Ub-AMC. 5. The fluorescence intensity (FI) was measured immediately with an Analyst HT reader using the kinetic settings. An excitation wavelength of 360 nm and emission wavelength of 425 nm are used with a dichroic mirror of 400 nm. The plate was read every 1'45" for 15 times. Data analysis: Assay data are analyzed using Bi | |
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