| Assay Method Information | |
| | HDAC Inhibition Assays |
| Description: | The HDAC 1, 2 assays employed buffer A, which contained 20 mM HEPES, pH 8.0, 1 mM MgCl2, 137 mM NaCl, 2.7 mM KCl, 0.05% BSA. The HDAC3/SMRT assay employed buffer B, consisting of 20 mM HEPES, pH 8.0, 1 mM MgCl2, 50 mM NaCl, 2.7 mM KCl, 0.05% BSA, 0.005% Tween 20, and 10 μM IP4. The HDAC6 assay employed buffer C, consisting of 20 mM HEPES, pH 8.0, 1 mM MgCl2, 137 mM NaCl, 2.7 mM KCl, 0.5 mM TCEP (Calbiochem) and 0.05% BSA. The HDAC8 assay employed buffer D, consisting of 20 mM HEPES, pH 8.0, 1 mM MgCl2, 100 mM NaCl, 20 mM KCl, 0.1% n-octyl-β-D-glucoside (Anatrace) and 0.05% BSA. All steps were performed at room temperature (23° C.). The assay was performed by pre-incubating serial dilutions of test compounds with the target HDAC prior to initiation with substrate. Each compound was titrated in a 10-point dose response, using a 1:3 fold dilution scheme, with 0.15 ul of solution added by ECH0555 to the plate, followed by the addition of 20 μl of the appropriate HDAC isoform diluted in appropriate assay buffer. The incubation was allowed to proceed for 3 hours, then the appropriate substrate diluted in assay buffer (final substrate concentration ˜Km) was added and the reaction allowed to proceed for 60 min. |
| Affinity data for this assay | |
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