| Assay Method Information | |
| | In-vitro HDAC Enzymatic Endpoint Assay |
| Description: | Purified HDAC1-9 (0.5~5 nM) were incubated with 2 μM carboxyfluorescein (FAM)-labeled acetylated peptide substrate A or B and test compound for 60 min at room temperature, in HDAC assay buffer that contained 50 mM HEPES (pH 7.4), 100 mM KCl, 0.01% BSA and 0.001% Tween-20. Reactions were terminated by the addition of the known non-selective HDAC inhibitor LBH-589 (panobinostat) with a final concentration of 1.4 μM. Acetylated peptide substrate and deacetylated peptide product were separated on a Caliper LabChip EZ Reader II equipped with a 12-sipper chip in ProfilerPro separation buffer and fluorescence intensity in the substrate and product peaks was determined and analyzed using EZ Reader software (Caliper Life Sciences; Hopkinton, MA). The separation of substrate and product was performed under the following optimized conditions: upstream voltage = −500 V, downstream voltage = −1500V and pressure = −1.3 psi. The reactions were performed in duplicate for e |
| Affinity data for this assay | |
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