The SIRT-Glo™ Assays and Screening Systems are single-reagent-addition, homogeneous, luminescent assays that measure the relative activity of the NAD+-dependent histone deacetylase (HDAC) class III enzymes (sirtuins; SIRTs) from purified enzyme sources. The assays use an acetylated, luminogenic peptide substrate that can be deacetylated by SIRT activities. Deacetylation of the peptide aminoluciferin substrate is measured using a coupled enzymatic system in which a protease in the Developer Reagent cleaves the deacetylated peptide from the aminoluciferin substrate, releasing aminoluciferin, which is quantified in a reaction using Ultra-Glo™ recombinant firefly luciferase. The assay reaction is typically complete within 15–45 minutes with no sample manipulation. The SIRT-mediated luminescent signal is persistent with a half-life of greater than 3 hours, allowing batch processing of multiwell plates. The SIRT-Glo™ Assay is broadly useful for NAD+-dependent Sirtuin enzymes.
Nicotinamide, included in the SIRT-Glo™ Screening Systems or available separately, is a known inhibitor of SIRTs and used as a positive control inhibitor. Nicotinamide is supplied at a concentration of 1M in SIRT-Glo™ Buffer.
The HeLa Nuclear Extract, included in the SIRT-Glo™ Screening Systems or available separately, may be used as an assay chemistry control. HeLa Nuclear Extract is supplied at a concentration of 5mg/ml.
The SIRT-Glo™ Control Substrate, only available separately, is a non-acetylated form of the SIRT-Glo™ Substrate with the same amino acid sequence and can be used with the SIRT-Glo™ Assays and Screening Systems to confirm true SIRT inhibition in secondary screens. The Control Substrate is supplied at a concentration of 10mM and is sufficient for 480 assays in 96-well plate format when combined with the SIRT-Glo™ Reagent prepared with components in the SIRT-Glo™ Assays or Screening Systems.
Features - Benefits
Simple Measurement of Deacetylating Activities: Use a single-reagent-addition, homogeneous, add-mix-measure protocol for easy implementation from benchtop to screening. Highly Sensitive: Achieve 10- to 100-fold higher sensitivity than comparable fluorescence methods. Fast Data Acquisition: Measure maximum signal in as little as 10-15 minutes with persistent glow-type steady-state signal.
Applications
Determining SIRT inhibitor or activator potency.
SIRT enzyme profiling.
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