The Epigenase™ Universal SIRT Activity/Inhibition Assay Kit (Fluorometric) is a complete set of optimized buffers and reagents for measuring the activity/inhibition of total SIRT enzymes using nuclear extracts or purified SIRT isoforms (SIRTs 1-7) from a broad range of species such as mammals, plants, fungi, and bacteria, in a variety of forms including, but not limited to cultured cells and fresh and frozen tissues. Detection of inhibition or activation of SIRTs is important in elucidating mechanisms of epigenetic regulation of gene activation and silencing and may benefit diagnostics and therapeutics of cancer or neurological diseases. The Epigenase™ Universal SIRT Activity/Inhibition Assay Kit (Fluorometric) provides the components to successfully achieve this, and has the following advantages:
- Strip microplate format makes the assay flexible and quick: manual or high throughput analysis can be completed within 3.5 hours.
- Unique kit composition enables background signals to be very low, which allows the assay to be accurate, sensitive, reliable, and consistent.
- Innovative fluorometric assay measures SIRT activity/inhibition by directly detecting SIRT-converted deacetylated products, rather than trypsin-based peptide cleavage, thus eliminating assay interference caused by DMSO and thiol-containing chemicals, trypsin, and cellular lysyl endipeptidases.
- Both cell/tissue extracts and purified SIRT enzymes can be used, which allows for the detection of inhibitory effects of SIRT inhibitor in vivo and in vitro.
- Novel assay principle allows high sensitivity to be achieved. The activity can be detected from as low as 1 ng of purified SIRT enzyme, which is about 10 fold higher than that obtained by trypsin-based peptide cleavage assays.
- A deacetylated histone standard is included, which allows for the specific activity of SIRTs to be quantified.
- Nicotinamide, a SIRT inhibitor as the positive inhibition control, and trichostatin A (TSA), an inhibitor of HDACI/II used to block HDAC activity, are both included.
Acetylation of the epsilon amino group of specific lysine residues contained in core histones is one of the most robust epigenetic marks and is essential for the regulation of multiple cellular processes. The acetylation of histone by histone acetyltransferases (HAT) seems to be of particular significance, as it is associated with active regions of the genome. In contrast, histone deacetylation by histone deacetylase (HDAC) leads to transcription repression. So far, at least 4 classes of HDACs have been identified. Class I HDACs include 1, 2, 3 and 8. Class II HDACs are comprised of 4, 5, 6, 7, 9 and 10. Class III enzymes, known as the sirtuins, require NAD+ cofactors and include SIRTs 1-7. Class IV enzymes, which contains only HDAC11, has features of both Class I and II.
Unlike other known protein deacetylases, which simply hydrolyzeacetyl-lysine residues, sirtuins catalyze a reaction that couples lysine deacetylation to NAD hydrolysis, yielding O-acetyl-ADP-ribose and nicotinamide. Sirtuins have been implicated in influencing aging and regulating transcription, apoptosis, and stress resistance, as well as energy efficiency and alertness during low-calorie situations. SIRTs are also involved in the development of human diseases including cancer, diabetes, and various neurological diseases. For example, SIRT1 was found to be overexpressed in prostate cancer. It was also observed that SIRTs protect neurons in Alzheimer's disease.
Principle & Procedure
The Epigenase™ Universal SIRT Activity/Inhibition Assay Kit (Fluorometric) contains all reagents necessary for the measurement of SIRT activity/inhibition. In this assay, an acetylated histone SIRT substrate is stably coated onto the microplate wells. Active SIRTs bind to the substrate and removes acetyl groups from the substrate. The SIRT-deacetylated products can be recognized with a specific antibody. The ratio or amount of deacetylated products, which is proportional to the enzyme activity, can then be fluorometrically measured by reading the fluorescence in a a fluorescent micorplate reader at 530 excitation and 590 emission. The activity of the SIRT enzyme is proportional to the RFU (relative fluorescence units) measured.
||Other Supplier's Kit
||Immunoaffinity-based end product direct measurement
||Endopeptidase cleavage-based indirect detection
||Excellent: detect from as low as 1 ng of enzyme
||Poor: detect from 5 ng enzyme
||>20 with very low background
||<5 with high background
|Interference by Chemical Reagents
||Yes, affected by many detergent and solvents such as DMSO and thiol-containing chemicals
|Assay accuracy with use of cell lysates
||Poor, due to cellular endipeptidase interference
|Amount of Purified Enzymes Required for Inhibitor Screening
||5 to 50 ng per assay point
||20 to 600 ng per assay point
|Amount of Substrate Required for Assay
||<0.5 µM per assay point
||>20 µM per assay point
|Reliability of in vivo enzyme inhibition assay
|Accuracy of Enzyme Inhibition Assay
||High: direct detection of amount change of end product; not affected by chemical reagents in the assay
||Low: indirect detection of endopeptidase cleaved peptide; severely affected by a variety of chemical reagents in the assay
Fig. 1. Schematic procedure of the Epigenase™ Universal SIRT Activity/Inhibition Assay Kit (Fluorometric).
Fig. 2. Demonstration of high sensitivity of a SIRT activity assay achieved by using recombinant SIRT1 with the Epigenase™ Universal SIRT Activity/Inhibition Assay Kit (Fluorometric).
Fig. 3. Illustrated standard curve generated with SIRT assay standard by using the Epigenase™ Universal SIRT Activity/Inhibition Assay Kit (Fluorometric).