| Solution Information | help | |
| Enzyme: | Caspase-3 | |
| inhibitor: | BDBM70843 | |
| substrate: | n/a | |
| Solution Type: | Aqueous | |
| pH at Preparation: | n/a | |
| Temp. Prep.: | n/a | |
| Comments: | Materials: 1) Z-DEVD-aminoluciferin (Promega # G8093) 2) Ultra-GloTM Luciferin Detection Reagent (LDR) Promega #V859B dissolved in 75mM Hepes, pH 7.8, 10mM MgSO4. 3) Activated Caspase-3 (28 -227 aa) - provided by Dr. Salvesen's laboratory. 4) Assay Buffer (75mM Hepes, pH 7.8, 4mM DTT, 1mM EDTA, 0.01% Tween 20, o.1% BSA) 5) Corning 1536-well white microtiter plates (Cat #3725) Procedure for dose-response Caspase-3 selectivity assay: 1) Using Labcyte Echo550, dispense 25 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 3 nM Caspase-3 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 the diluted Z-DEVD-aminoluciferine substrate (1:2.67 v/v 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 Envis | |
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