The SIRT-Glo™ Assay is a single-reagent-addition, homogeneous, luminescent assay that measures the relative activity of the NAD+-dependent histone deacetylase (HDAC) class III enzymes (sirtuins; SIRTs) from purified enzyme sources. The assay uses 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.
- Use a single-reagent-addition, homogeneous, add-mix-measure protocol for easy implementation from benchtop to screening.
- Achieve 10- to 100-fold higher sensitivity than comparable fluorescence methods.
- Measure maximum signal in as little as 10–15 minutes with persistent glow-type steady-state signal.