| Assay Method Information | |
| | Inhibitory Activity of the Compounds on HDAC6 |
| Description: | Prepare a 10 mM stock solution of a compound with DMSO. Take 10 μl of stock solution and dilute with 90 μl DMSO to become a 1 mM working solution. The compound was three-fold serially diluted to total of 11 concentrations including a DMSO negative control. Add 3 μl of each concentration to 197 μl reaction buffer (20 mM Hepes pH 8.0, 137 mM NaCl, 2.7 mM KCl, 1 mM MgCl2, 0.05% BSA, 0.5 mM TCEP), mix well and add 10 μl to a 384-well plate. In the final reaction system, the concentrations of the compound were 10 μM to 0.51 nM. Duplicate, and add 10 μl 3XHDAC solution (BPS, Cat. 50006, 0.3 nM) to each well, followed by incubating at 23° C. for 20 minutes. Then, add 3× substrate solution (Anaspec, Cat. 61855, 15 μM), centrifuge to mix, and incubate at 23° C. for 90 minutes. Add 30 μl trypsin/SAHA mixture (20 mM Hepes pH 8.0, 100 mM NaCl, 10 mM SAHA, 0.01 mg/ml trypsin), incubate at 23° C. for 60 minutes to terminate the reaction. Finally, read fluorescence data by Envision (390 nm excitation, 460 nm emission). A high value at 430 nm indicates high kinase activity, while low value at 430 nm indicates that the kinase activity was inhibited. Finally, analyze the data by XLfit5 software and calculate the IC50 value of the compound. Vorinostat (SAHA) was a positive reference compound. |
| Affinity data for this assay | |
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