HDAC Assays
Target-specific Assays

Histone Deacetylase (HDAC) Assay Services

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

Each HDAC assay and Sirtuin assay is optimized based on its specific substrate.

  • Direct measurement of histone deacetylase activity
  • Low scale, large scale screening, and high-throughput options
  • All HDAC and sirtuin enzymes are produced in-house and available for purchase.

In addition to biochemical assays, we also offer cell-based HDAC assays to measure enzyme activity via the qualification of acetylated histone or tubulin. The assays can be performed via HDAC-Glo, ELISA, or Western blotting.

For drug discovery, the offering comprises all needs of compound screening: primary biochemical HDAC assays, cell-based secondary histone deacetylase assays together with the option of performing HDAC screening via biophysical methods for mechanistic and binding studies.

Histone deacetylase screening is performed at our US facility. A business development manager in your area will be happy to talk to you about your research needs and find out how our team of scientists can support your project best.

HDAC Assay Details

  • Assay format
  • Assay principle
  • Assay setup
  • Screening schedule
  • Cell-based HDAC testing
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, 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 10: acetylated polyamine: Ac-Spermidine-AMC
  • HDAC 4, 5, 7, 9, 11: Trifluoroacetyl lysine
  • Sirt 5: Ac-Lys(Succinyl)-AMC
Assay principle

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

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.

Screening schedule

Reaction Biology performs screening on the full HDAC panel on a bi-weekly basis.

Please see the upcoming screening dates here.

Cell-based HDAC testing

Reaction Biology offers two assay formats for HDAC screening in intact cells:

1. Testing of the enzymatic activity of HDAC targets via quantification of deacetylation of substrates.

2. Binding of a compound to the HDAC target protein performed with the NanoBRET Target Engagement Intracellular HDAC Assay.

More information can be found on our Cell-based Epigenetic Assay webpage.