| Assay Method Information | |
| | HDAC Enzyme Assay |
| Description: | The pan-HDAC in vitro activity is detected by HDAC-Glo™/II Assay and Screening System (#G6420, Promega, WI, USA). This system is single-reagent-addition, homogeneous, luminescent assays that measure the relative activity of histone deacetylase (HDAC) class I and II enzymes from cells, extracts or purified enzyme sources. The assay procedure is as follows: 1. Seed cells in 100 mL culture medium with 10000 cells/well in white 96-well culture plate (Corning) and incubate overnight. 2. Prepare test compounds and SAHA stock in DMSO and dilute stock to ten-fold of working concentration by HDAC-Glo™ I/II Buffer (Provided in HDAC-Glo™ I/II Assay and Screening System). Use 10% DMSO in HDAC-Glo™ I/II Buffer as non-inhibitor control. 3. Remove medium in white 96-well plate, wash cells with PBS, and add 90 mL fresh serum free medium to wells. 4. 10 mL of non-inhibitor control and ten-fold working concentration compounds were directly added to the wells and incubate for 2 hours at 37° C. 5. Add 25 mL substrate (HDAC-Glo substrate in HDAC-Glo™ I/II Buffer+0.1% Developer reagent+1% Triton X-100) to wells and the chemiluminescence signal was recorded every 5 min over a period of 60 mins in a SpectraMax i3 Multiplate reader (Molecular Devices, Sunnyvale, Calif., USA) for 1 h. The activities are expressed in percentage of non-inhibitor control minus blank control. |
| Affinity data for this assay | |
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