| Assay Method Information | |
| | HDAC6 and HDAC8 Inhibitory Activity Assay |
| Description: | 2×HDAC enzymes were dispensed in each well of the reaction plate except for the control well without HDAC enzyme, and buffer was dispensed in the control well without the enzyme. Using acoustic technology (Echo550, nanoliter range), compounds dissolved in 100% DMSO were added to the enzyme mixture, spun down, and pre-cultured at room temperature for 10 minutes. All reaction wells were placed with a 2× substrate mixture (fluorogenic HDAC substrate) to initiate the reaction and then spun down. Culture was performed at 30° C. for 1 hour for HDAC6 and 2 hours for HDAC8. Afterwards, a developer was added along with trichostatin A to halt the reaction and develop fluorescence. Dynamics measurements were performed with Envision (Ex/Em=360/460 nm) for 20 minutes with 5-minute intervals, and values from endpoint reading were taken for analysis after the phenomenon reached a plateau. |
| Affinity data for this assay | |
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