SIRT 1 kit

The HTRF SIRT1 assay enables a straigthforward screening and characterization of compounds activating or inhibiting SIRT1 activity
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  • Rapid
  • Low enzyme consumption
The HTRF SIRT1 assay enables a straigthforward screening and characterization of compounds activating or inhibiting SIRT1 activity
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Overview

SIRT1 belongs to class III of the silent information regulator 2 (Sir2) family of proteins, which are histone/protein deacetylases (sirtuins). The use of SIRT1 modulators has been described as a potential therapeutic solution for treating multiple disease states including cancer, diabetes, aging, and neurodegenerative, metabolic or cardiovascular diseases. This rapid, simple assay enables the discrimination of polyphenolic compounds and can be used for high throughput screening to identify both inhibitors and activators of SIRT1 activity.

Assay principle

The HTRF SIRT1 assay uses an anti-acetyl specific MAb labeled with Eu-cryptate and an acetylated-d2 labeled substrate. The maximum signal is obtained in absence of SIRT1 and decreases proportionally to the deacetylation process. The quantity of deacetylated peptides is then directly correlated to the signal extinction.

Assay protocol

The assay consists of two steps: an enzymatic step followed by a universal detection step. The SIRT1 enzyme is first incubated in the presence or absence of compounds with the acetylated d2 labeled substrate. NAD+ is added to start the reaction. The Eu-cryptate labeled MAb is then added. The detection buffer contains EDTA and a SIRT inhibitor (nicotinamide) to stop the enzymatic detection.

Screening Robustness

Z' values for different SIRT1 concentrations in twenty-four replicates were run using 4 selected SIRT1 concentrations: 0 mU of enzyme (no deacetylation), 100 mU of enzyme (100% deacetylation), 10 mU and 20 mU corresponding to the EC50 and EC80 respectively.

Finding activator and inhibitor compounds

Titration curves were performed using 3 classes of compounds: two polyphenolic compounds (resveratrol and piceatannol), two non-polyphenolic compounds (activator1 and activator2 and one reference inhibitor (nicotinamide)

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