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Assay Method Information |
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| | Inhibition Assay |
| Description: | Human recombinant HDAC4 was expressed in full length form (aa 2-1084) in Sf9 insect cells (obtained from ATCC) using baculovirus generated with Bac-to-Bac system (Invitrogen). Test compounds were serially diluted to reach final test concentrations from 0.003 uM to 100 uM. HDAC4 and test compounds were incubated in 25 mM Tris buffer pH 8.0 containing 137 mM NaCl, 2.7 mM KCl, 1 mM MgCl2 and 0.05% (w/v) bovine serum albumine for 2 hours at room temperature in presence of 10 uM of acetyl-Gly-Ala-Lys(E-trifluoroacetyl)-AMC (AMC=7-amino-4-methyl coumarin) in a final volume of 200 ul. Control wells with HDAC4 only (positive control) and without HDAC4 (negative control) were included on the microplate. Bovine trypsin (10 ul of a 0.4 mg/ml solution) was added and the plate incubated for additional 15 minutes at room temperature. The plate was placed in a fluorescence microplate reader, and read at an excitation wavelength of 360 nm and an emission wavelength of 450 nm. |
| Affinity data for this assay | |
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