| Assay Method Information | |
| | Fluorescence-Based Deacetylation Assay |
| Description: | Deacetylation assays using Boc(Ac)Lys-7-amino-4-methyl-coumarin (MAL; Bachem, Bubendorf, Switzerland) as a substrate were performed in a volume of 20 μl in 384-well low volume plates (Eppendorf) in a reaction buffer containing 25 mM Tris-HCl (pH 8.0), 137 mM NaCl, 1 mM MgCl2, 2.7 mM KCl, 1 mg/ml BSA, and 1mM DTT. Enzymes were added at different concentrations to wells in a volume of 10 μl and were preincubated with inhibitors (volume 1 μl, diluted in dimethyl sulfoxide) or with dimethyl sulfoxide as a control for 10 min at room temperature. Subsequently, 10 μl 2× concentrated substrate solution containing 200 μM MAL and 2 mM NAD+ was added to initiate the reaction, which was incubated at 37 °C for 4 h. This allowed for ~50% deacetylation of MAL. After incubation, 20 μl of trypsin solution (0.5 mg/ml) was added, and the trypsin cleavage reaction was allowed to proceed at 37 °C for 1 h. Fluorescence readings were obtained using a fluorescence reader(GENiosPro TECAN), with the excitation wavelength set to 360 nm and the emission set to 465 nm. |
| Affinity data for this assay | |
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