Histone deacetylase activity of HDACs was measured using a fluorometric histone deacetylase kit (CS1010; Sigma-Aldrich) following the manufacturer’s instructions. Equal volumes of purified HDAC-1 beads obtained from immunoprecipitates from cells expressing HDAC-1-Flag as described above were used. For the HDAC assay, 30 μL of assay buffer were added to 100 μmol/L HDAC substrate and 20 μL of washed HDAC-1 beads. After 30 minutes of incubation at 30 °C, 10 μL of the developer solution was added, allowing the fluorescent reporter to be released from the deacetylated substrate. Fluorescence was detected using a Cytation image plate reader (BioTeK) with excitation at 355 nm and emission at 460 nm. Measurements were taken from 4 different biological replicates and normalized to the amount of HDAC-Flag present in each sample, as determined by quantification of a 30-μL sample subjected to Western blotting.
Fluorometric histone deacetylase kit
Manufactured by Merck Group
The Fluorometric histone deacetylase kit is a laboratory tool used to measure the activity of histone deacetylase enzymes. It provides a quantitative assessment of histone deacetylase activity in cell lysates or purified enzyme samples.
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2 protocols using fluorometric histone deacetylase kit
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Fluorometric Assay of HDAC Activity
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Fluorometric HDAC Activity Assay
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HDAC activity was measured with a fluorometric histone deacetylase kit (Sigma-Aldrich) as described previously (Du et al., 2014) . Briefly, stock solutions of HDT-NTDs were diluted into the provided assay buffer containing the HDAC substrate to a final concentration of 100 nM in 100-μL volume. Then, the assay reaction was incubated at 30°C for 30 min, followed by adding 10 μL of the developer solution. The developer allows the release of the fluorescent reporter upon deacetylation of the substrate. A Varioskan Flash plate reader (Thermo Scientific) with an excitation at 355 nm and emission at 460 nm was used for measuring fluorescence. Triplicate measurements were taken, and the readings were normalized.
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