Activity of HDAC was determined using an HDAC assay kit (Active Motif, Carlsbad, CA, USA). Briefly, 30 μL nuclear extracts, which were extracted from cell samples treated for 12 h with the Qproteome Nuclear Protein kit (Qiagen), were incubated with 1 mM HDAC substrate in the assay buffer in a total volume of 50 μL for 1 h at 37°C, followed by termination of the reaction by addition of 50 μL HDAC developing solution. The fluorescence intensity was measured at a wavelength of 405 nm.
Hdac assay kit
Manufactured by Active Motif
Sourced in United States, Belgium
The HDAC assay kit is a laboratory tool designed to measure the activity of histone deacetylase (HDAC) enzymes. It provides a quantitative assessment of HDAC activity in biological samples.
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Lab products found in correlation
Anti hdac3 antibody, by Abcam (2 mentions)
Synergy ht, by Agilent Technologies (2 mentions)
Qproteome nuclear protein kit, by Qiagen (1 mentions)
Epoch colorimetric plate reader, by Agilent Technologies (1 mentions)
Victor x5 plate reader, by PerkinElmer (1 mentions)
Ne per nuclear and cytoplasmic extraction, by Thermo Fisher Scientific (1 mentions)
Cholesterol quantitation kit, by Merck Group (1 mentions)
Free fatty acid quantitation kit, by Merck Group (1 mentions)
10 protocols using hdac assay kit
1
Quantifying HDAC Activity in Nuclear Extracts
2
HDAC Activity Quantification in Nuclear Extracts
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HDAC activity was measured with 1.545 μg of nuclear protein extract following the manufacturers protocol (HDAC Assay Kit, colorimetric, Active Motif, Carlsbad, CA). As suggested in the protocol for samples with potentially low HDACs, we extended the initial HDAC reaction time to three hours. Since the kit was developed based on nuclear extracts from mammalian cells, we envisioned that extending the incubation time will help complete the deacetylation reaction. Samples were measured in triplicate (standards were measured in duplicate) using EPOCH colorimetric plate reader (Biotek, Winooski, VT) at 405 nm. In the assay reaction, a short peptide substrate was added along with the nuclear extract and other reagents as per the protocol. This substrate contains acetylated lysine residues and can be deacetylated by most HDAC enzymes. Active HDACs from the experimental samples would then bind to the added substrate by removing acetyl groups from the substrate. This reaction then yielded an HDAC-deacetylated colored product, which was measured by the colorimetric plate reader. The amount of deacetylated product in the reaction is directly proportional to the amount of active HDAC enzymes present in our samples [37 (link)].
3
Measuring HDAC Activity in Cardiac Myocytes
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HDAC activity was assessed using the HDAC assay kit (Active Motif) according to the manufacturer's instructions. In brief, 10 μg of cardiac myocyte lysate in the presence or absence of 1 μM SAHA was added to each well in 96-well microtiter plates with HDAC substrate, provided by the assay kit, at 37°C for 1 h. The optical density of each well was measured at 405 nm.
4
Measuring HDAC3 Deacetylase Activity
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HDAC3 deacetylase activity was measured with the HDAC Assay Kit (Active Motif 56200) according to manufacturer’s instructions on immunoprecipitated HDAC3 (as described above) from control, MHD3KO, Y298F, or WT-rescue BMDM with 1mg of starting material using anti-HDAC3 antibody (Abcam 7030). Substrate solution containing short peptides with acetylated lysine residues were added directly to the HDAC3-antibody-Protein A bead slurry complex. After incubation with developing solution, fluorescent signals from standards and samples were detected by a plate reader system (Biotek Synergy HT), and deacetylase activity was determined by the standard curve method.
5
HDAC Activity Quantification in Nuclei
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The nuclear proteins were extracted as mentioned in Immunoblot analysis. The nuclear HDAC activity was measured in biological triplicates using HDAC Assay Kit (Active Motif, .56200), which is a biological assay to determine the HDAC activity using a short peptide substrate that contains an acetylated lysine residue. The HDAC activity is determined as the concentration of active HDAC by making a standard curve.
6
HDAC Activity Assay with Inositol Phosphates
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HDAC activity assays carried out as described in (10 (link)). To perform the titration, 62 nM protein was incubated with varying concentrations of inositol-1,4,5,6-tetraphosphate and inositol-1,4,5-triphosphate for 30 min at 37°C then the HDAC activity was measured using the HDAC Assay Kit (Active Motif) and read using a Victor X5 plate reader (PerkinElmer). Experiments were performed in triplicate and data were analyzed using GraphPad Prism (version 4.0, GraphPad Software, Inc.).
7
Measuring HDAC3 Deacetylase Activity
8
HDAC Enzyme Activity in Alcohol-Treated MDDCs
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Normal MDDCs were treated for 24 hrs. with 0.1 and 0.2% alcohol (n = 3) followed by NE-PER nuclear and cytoplasmic extraction (Thermo Scientific, Cat. #78835). Whole cell lysate extraction was performed for MDDCs from alcohol users (n = 6) and controls (n = 6). HDAC enzyme activity was measured with the HDAC assay kit following manufacture's protocol (Active Motif, Cat. #102209) and as previously described [14 (link),22 ]. HDAC enzyme activity was calculated across the groups and graph as pmoles/min/mg of protein. The fluorescence was measured in a Biotek plate reader (Winooski, VT), 360 nm excitation and 460 nm emission.
9
HDAC Activity Determination Protocol
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Determination of HDACi activity was performed with HDAC assay kit (Active Motif, La Hulpe, Belgium) as described by the manufacturer with an incubation time of 2 h at 37°C, using 6-PN and 8-PN at rising concentrations (5, 10, 20, 50 and 100 µmol/L, both from Sigma-Aldrich).
10
Quantitation and Assays for Metabolic Markers
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The primers for gene expression and ChIP were designed by Primer 3 software (v. 0.4.0) or obtained from previous publications: Dbp E-box ChIP primers are based on Ripperger and Schibler (2006) , and Nampt E-box chip primers are based on Nakahata et al. (2009) . All the primer sequences are reported in Table S7.
b-Hydroxyl-Butyrate Quantitation Fresh blood was centrifuged at 1500 g for 15 min and serum isolated. b-Hydroxyl-butyrate level were quantified using b-Hydroxybutyrate LiquiColor Test (Endpoint) (StanBio Cat. N. 2440-058) according to the manufacturer's instructions. For b-Hydroxybutyrate levels in intestine and liver, we used Abcam BHB Assay kit (Cat. N. ab83390). The samples were prepared and deproteinated with PCA according to the manufacturer's instruction.
Fatty Acid and Cholesterol Quantitation 10-15 mg of liver or intestinal tissue were used to check free fatty acid and total cholesterol levels. Free fatty acid were quantified using the ''Free fatty acid quantitation kit'' (Sigma-Aldrich Cat. N. MAK044) according to manufacturer's instructions. Total cholesterol was quantified using the ''Cholesterol quantitation kit'' (Sigma-Aldrich Cat. N. MAK043) according to manufacturer's instructions.
HDAC Activity Assay 15 mg of intestinal or liver nuclear extract was tested using HDAC assay kit (Active Motif Cat. N. 56200) according to manufacturer's instructions.
b-Hydroxyl-Butyrate Quantitation Fresh blood was centrifuged at 1500 g for 15 min and serum isolated. b-Hydroxyl-butyrate level were quantified using b-Hydroxybutyrate LiquiColor Test (Endpoint) (StanBio Cat. N. 2440-058) according to the manufacturer's instructions. For b-Hydroxybutyrate levels in intestine and liver, we used Abcam BHB Assay kit (Cat. N. ab83390). The samples were prepared and deproteinated with PCA according to the manufacturer's instruction.
Fatty Acid and Cholesterol Quantitation 10-15 mg of liver or intestinal tissue were used to check free fatty acid and total cholesterol levels. Free fatty acid were quantified using the ''Free fatty acid quantitation kit'' (Sigma-Aldrich Cat. N. MAK044) according to manufacturer's instructions. Total cholesterol was quantified using the ''Cholesterol quantitation kit'' (Sigma-Aldrich Cat. N. MAK043) according to manufacturer's instructions.
HDAC Activity Assay 15 mg of intestinal or liver nuclear extract was tested using HDAC assay kit (Active Motif Cat. N. 56200) according to manufacturer's instructions.
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