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| Solution Information | help | |
| Enzyme: | Sentrin-specific protease 8 | |
| inhibitor: | BDBM33185 | |
| substrate: | n/a | |
| Solution Type: | Aqueous | |
| pH at Preparation: | n/a | |
| Temp. Prep.: | n/a | |
| Comments: | Materials: 1) Z-RLRGG-aminoluciferin (Promega #X554X) 2) Ultra-GloTM 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 counterscreen validation assay 1) Using Labcyte Echo550, dispense 25 nL of serially diluted compounds to columns 5-48 and DMSO to columns 1-4. 2) In a separate tube, prepare the Reaction Mixture, by combining equal volumes of the Enzyme Mix (160 nM SENP8 in the assay buffer with 10 mM ATP) and Substrate Mix (40 uM Z-RLRGG-aminoluciferine in LDR). Incubate for 60 min at room T. 3) Using Thermo Scientific MultiDrop Combi nL, dispense 2 ul of Reaction Mixture into columns 3 through 48, while 1 ul of just Assay Buffer combined with the 1 uL of the Substrate Mix into columns 1 and 2. 4) Centrifuge plates br | |
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