Sirtuins (SIRT) are enzymes that play a crucial role in various cellular processes including metabolism and stress response. SIRT functions as a NAD⁺-dependent deacetylase, which removes acetyl groups from proteins and alters their activity.
 
The SIRT Activity Assay kit can measure the activity of SIRT using a FRET-based peptide substrate. The peptide has a fluorophore on the N-terminus, quencher on the C-terminus, and an acetylated lysine side chain. Before the peptide is cleaved, the quencher prevents fluorescence from being emitted. When a reaction mixture containing the SIRT enzyme and a Lys-protease is added to the peptide substrate, SIRT first deacetylates the acetyl group on the lysine. Then, once the lysine is exposed, the Lys-protease will cut the peptide on the carboxyl side of the lysine, splitting the peptide in half and separating the fluorophore from the quencher. Once the fluorophore is separated from the quencher, it will emit fluorescence that can be measured at Ex/Em: 350/460 nm.