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| | HDAC Enzyme Assay |
| Description: | Fluorescence readings were carried out on a CytofluorR Series 4000 fluorescence multiwell plate reader (Perspective Biosystems). Stock solutions of the HDAC inhibitor (10 mM) and substrates (10 mM) were freshly prepared in DMSO. The buffer for all experiments was 25 mM Tris/Cl (pH 8.0), 137 mM NaCl, 2.7 M KCl, and 1 mM MgCl2. To avoid loss of enzyme activity through repeated freeze/thaw cycles, aliquots of HDAC1 and HDAC6 were prepared and stored at -80 °C, and recombinant HDAC7 enzyme was freshly prepared. The enzyme was diluted with buffer to a final concentration of 0.005 ng/ul, and enzyme assays were carried out in 50-ul reaction volumes. Developer solution was used as described for HDAC1 by the supplier, and was added after 30-min incubation at 37 °C. The final substrate concentration was 50 uM. Bovine serum albumin was used at 100 ug/ml. |
| Affinity data for this assay | |
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