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Epigenase Universal SIRT Activity/Inhibition Assay Kit (Colorimetric)

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Suggested Workflow
Nuclear Protein Extraction
 
 
HDAC Activity/Inhibition Assay
 
Schematic procedure of the Epigenase™ Universal SIRT Activity/Inhibition Assay Kit (Colorimetric).
Demonstration of high sensitivity of a SIRT activity assay achieved by using recombinant SIRT1 with the Epigenase™ Universal SIRT Activity/Inhibition Assay Kit (Colorimetric).
Illustrated standard curve generated with SIRT assay standard by using the Epigenase™ Universal SIRT Activity/Inhibition Assay Kit (Colorimetric).
Input Type: Nuclear Extracts, Purified Enzyme
Research Area: Histone Acetylation
Target Application: Activity Measurement
Vessel Format: 96-Well Plate
100% Guarantee: 6 months
Catalog No.SizePriceQty
P-4036-4848 assays $221.00 
P-4036-9696 assays $399.00 
Availability: Usually Ships In 1-2 Days 
Product Overview

The Epigenase™ Universal SIRT Activity/Inhibition Assay Kit (Colorimetric) 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 (Colorimetric) 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 colorimetric 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.

Background Information
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 (Colorimetric) 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 colorimetrically measured by reading the absorbance in a microplate spectrophotometer at 450 nm. The activity of the SIRT enzyme is proportional to the OD intensity measured.

  Epigenase™ Kit Other Supplier's Kit
Assay Principle Immunoaffinity-based end product direct measurement Endopeptidase cleavage-based indirect detection
Assay Format 96-Well Microplate-based 96-Well Microplate-based
Sensitivity Excellent: detect from as low as 1 ng of enzyme Poor: detect from 5 ng enzyme
Signal-Noise Ratio >20 with very low background <5 with high background
Interference by Chemical Reagents No Yes, affected by many detergent and solvents such as DMSO and thiol-containing chemicals
Assay accuracy with use of cell lysates Excellent 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 Excellent Poor
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 (Colorimetric).

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 (Colorimetric).

Fig. 3. Illustrated standard curve generated with SIRT assay standard by using the Epigenase™ Universal SIRT Activity/Inhibition Assay Kit (Colorimetric).

Product Components

WB (10X Wash Buffer)
HO1 (SIRT Assay Buffer)
HO2 (SIRT Substrate)
HO3 (SIRT Assay Standard, 50X)
HO4 (Capture Antibody, 1000X)
HO5 (Detection Antibody, 2000X)
NAD (SIRT Co-factor, 50X)
TSA (HDAC Inhibitor, 50 µM)
NAM (SIRT Inhibitor,  50 µM)
DS (Developer Solution)
SS (Stop Solution)
8-Well Assay Strips (With Frame)
Adhesive Covering Film
User Guide

User Guide & MSDS

[User Guide]*
*Always use the actual User Guide that shipped with your product. Is the above file locked? You can also request user guides by emailing info@epigentek.com along with your contact information and institution name.

Customer Reviews

Rating By j***@forgelifescience.com Verified Purchase Reviewed on: Wednesday 28 December, 2016
Application Description
The product was being tested for the potential continued usage of inhibitor/activator screening of various sirtuin enzymes.

Pros: Overall the kit is a positive assay to test sirtuin activity. The lack of fluorophore and second digestion step are both positives as both components have been shown to have potential interference in sirtuin activity.

Cons: As the kit is overall simple to use, it does hold limitations as the user is restricted to the provided peptide sequence and the acetyl modification on the peptide. Sirtuins 1 – 7 are known to have various enzymatic activity or efficiency to various peptide substrates and modifications (acetyl, myristoyl, succinyl, etc) Some sirtuin activity against lysine-acetyl modifications is undetectable by any method except for HPLC and would not be detected using this kit. I have not performed enough tests or research to confirm the sensitivity of all seven sirtuins against an acetyl peptide in this particular kit. Additionally, having no knowledge of the peptide concentrations prevents the generation of any enzyme kinetic data, such as Km or Kcat.

Having a large number of reagents, especially antibodies, leads to the greater potential of one of the reagents going “off” and in turn leading to greater number of trouble shooting.

Other Thoughts
The user guide was long and detailed which can be a positive and negative. The protocol itself is initially confusing due to the individual reagents are identified as a code name which requires the user to flip back and forth between the protocol and the component list. A few sections for example, “Materials Required But Not Supplied” and “General Product Information” could be eliminated or reduced in size to compress the assay protocol. The “A Brief Overview” section can be reduced to the last paragraph and bullet points. Anyone buying this kit is most likely well aware of the biological significance of sirtuins and in turn is why they are buying this kit. The protocol itself in keeping with the times should be more picture oriented than text to make for ease of use.

The box and presentation of the product was done well and the reagent vials were clearly labeled.

Overall the usage of the kit was very positive, but would not fit our experimental needs for the high throughput screening of sirtuin modulators due to the overall cost of a 96-well assay and to a smaller extend the number of protocol steps.
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