| Solution Information | help | |
| Enzyme: | Sentrin-specific protease 8 | |
| inhibitor: | BDBM32030 | |
| substrate: | n/a | |
| Solution Type: | Aqueous | |
| pH at Preparation: | n/a | |
| Temp. Prep.: | n/a | |
| Comments: | Materials: 1) Z-RLRGG-aminoluciferin (Promega #X554X) 2) Luciferin Detection Reagent (LDR) Promega #V859B dissolved in 75mM Hepes, pH 7.8, 10mM MgSO4. 3) Full-length SENP8 - provided by Dr. Salvesen's laboratory. 4) Assay Buffer (75mM Hepes, pH 7.8, 4mM DTT, 1mM EDTA, 0.01% Tween 20) 5) Corning 1536-well white microtiter plates (Cat #3725) Procedure for dose-response SENP8 assay: 1) Using Labcyte Echo550, dispense 20 nL of serially diluted compounds to columns 5-48 and DMSO to columns 1-4. 2) Using Thermo Scientific MultiDrop Combi nL, dispense 1 ul of 160 nM SENP8 in Assay Buffer into columns 3 through 48, while 1 ul of just Assay Buffer into columns 1 and 2. 3) Using Thermo Scientific MultiDrop Combi nL, dispense 1 ul of 80 uM Z-RLRGG-aminoluciferine in LDR to all wells of the plate. 4) Centrifuge plates briefly 5) Incubate at room temperature for 60 min. 6) Read plates on a Perkin Elmer Envision 2104 plate reader using the ultra-sensitive luminescence mode. | |
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