| Assay Method Information | |
| | HDAC6 Enzymatic Activity Assay |
| Description: | Dose response testing of HDAC6 and HDAC 1 were run at Reaction Biology Corporation (RBC). Inhibition of HDAC enzymes was performed using N-terminal GST tagged human full-length recombinant HDAC6 (H88-30G, SignalChem) and C-terminal FLAG His tag human full-length recombinant HDAC1 produced in insect cells (KDA-21-365, RBC). Enzyme reactions were run in 50 mM Tris-HCl, pH8.0, 137 mM NaCl, 2.7 mM KCl, and 1 mM MgCl2 with 1 mg/ml BSA, 1% DMSO freshly added. 2× enzyme was delivered in the wells of the reaction plate except for the “no enzyme” control wells. In the latter, buffer was added. Compounds in 100% DMSO were delved into the enzyme mixture by acoustic technology (Echo550). Plates are spun down and compounds were incubation with the enzyme for 10 min at room temperature. 2× Fluorogenic peptide from p53 residues 379-382 (RHKKAc, 10 μM final concentration) was added in all wells to initiate the reaction, followed by 1 h incubation at 30° C. Developer containing Trichostatin A was added to stop the reaction and generate fluorescent color. A kinetic measurement was carried out for 20 min with 5 min interval on the Envision plate reader (Perkin Elmer, Ex/Em=360/460). End point reading i.e., plateau of the development reaction was used for analysis. Data was reported by RBC as percentage enzyme activity. Percentage inhibition was calculated by subtracting percentage enzyme activity from 100. IC50 values were calculated using GraphPad Prism 9 based on the log(inhibitor) vs. response—Variable slope (four parameters) equation. |
| Affinity data for this assay | |
|---|---|
| If you find an error in this entry please send us an E-mail | |