HDAC Assay (Histone Deacetylase Assay) and Sirtuin Assay Services

Reaction Biology offers Zn2+-dependent HDAC assays and NAD+-dependent sirtuin assays for compound screening with fluorescence-based activity measurement.

Each assay is optimized based on its specific substrate.

  • Direct measurement of enzyme activity
  • Low scale, large scale screening and high-throughput options as well as hit to lead screening options are available
  • Please ask us for assay development and research collaboration options
  • All HDAC and sirtuin enzymes are produced in house and available for purchase

List of Targets

Target Synonyms Data
HDAC1 HDAC1 Data sheet
HDAC10 HDAC10
HDAC11 HDAC11
HDAC2 HDAC2 Data sheet
HDAC3 HDAC3 Data sheet
HDAC4-Class2a sub HDAC4-Class2a Data sheet
HDAC5-Class2a sub HDAC5-Class2a Data sheet
HDAC6 HDAC6 Data sheet
HDAC7-Class2a sub HDAC7-Class2a Data sheet
HDAC8 HDAC8 Data sheet
HDAC9-Class2a sub HDAC9-Class2a Data sheet
Nuclear Extract Nuclear Extract Data sheet
SIRT1 SIRT1 Data sheet
SIRT2 SIRT2 Data sheet
SIRT3 SIRT3 Data sheet
SIRT5 SIRT5 Data sheet

Assay Details

Assay format

Fluorogenic peptides that used to measure the enzymatic activity of HDAC and Sirtuins are listed below. We have established assays with substrates specific to the individual enzymes:

  • HDAC 1, 2, 3, 6, 10, and Sirt 1, 2, 3: acetylated tetrapeptide comprising p53 residues 379-382: RHKKAc-AMC
  • HDAC 8: twice acetylated tetrapeptide comprising p53 residues 379-382: RHKAcKAc-AMC
  • HDAC 4, 5, 7, 9, 11: Trifluoroacetyl lysine
  • Sirt 5: Ac-Lys(Succinyl)-AMC
Assay principle

HDAC_assy_principle

The hydrolytic deacetylation reaction catalyzed by classes I, II, and IV HDACs on a nucleosome is shown.

HDAC_assy_principle

The structure of a synthetic HDAC substrate used for protease-coupled assays is shown. The moiety required for signal generation is linked to the carboxyl of the acetyllysine that is the target for deacetylation by the HDAC. After deacetylation by the HDAC, the fluorescent signaling group 7-amino-4-methyl coumarin (AMC) can be quantified.

Assay setup

Setups: Single-dose screening in duplicates or IC50 value determination with 5 or 10 concentrations. Other screening formats are available upon request.

Controls: No inhibitor (DMSO vehicle) control and for every target, one target-specific control compound is tested in 10-dose IC50 format. 

Turnaround time: 10 business days for standard projects. Expedited scheduling and data delivery can be arranged prior to the commencement of the studies.

Report: The raw data, % enzyme activity and control compound IC50 values will be reported in Excel format for single-dose assays. For IC50 orders, raw data, IC50 values, and curve fitting will be delivered in Excel format. Assay conditions, target, and substrate information are available upon request. Requirements for this information should be noted prior to the commencement of the study.

Screening facility: This assay is performed at our screening facility in Malvern, PA, USA.

Compound requirements: In brief, for a standard project, 20 µl of a 10 mM DMSO stock or solid material is needed. Less material is needed for large scale screening. Please refer to our FAQs for information regarding compound preparation and shipping.