Human SIRT1 assay

SIRT1 belongs to class III of the silent information regulator 2 (Sir2) family of proteins, which are NAD+-dependant histone/protein deacetylases (sirtuins). The use of SIRT1 modulators has been described as an attractive 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 Eu3-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.The assay consists of two steps: an enzymatic step followed by a universal detection step.

Enzymatic step

The SIRT1 enzyme is incubated in the presence or absence of compounds with the acetylated d2 labeled substrate. NAD+ is added to start the reaction.

Detection step

The Eu3+ Cryptate labeled MAb is added. The detection buffer contains EDTA and a SIRT inhibitor (nicotinamide) to stop the enzymatic detection.

Product Performance

Screening Robustness

Z' values for different SIRT1 concentrations in twenty-four replicates were run using 4 selected SIRT1 concentrations: 0mU of enzyme (no deacetylation),100mU of enzyme (100% deacetylation), 10mU And 20mU 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
  • One inhibitor of reference: nicotinamide

Part#, inserts & MSDS

Ordering Info

DescriptionCat. noProduct insertMSDS
SIRT 1 kit - 1,000 tests64SI1PEB
SIRT 1 kit - 20,000 tests64SI1PEC

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