List of HDAC and Sirtuin Assays
|Nuclear Extract||Nuclear Extract||Data sheet|
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
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.
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.