A Sensitive Fluorometric Assay for Screening SIRT5 Inhibitors
- Easy-to-use kits (two-step) for screening SIRT5 inhibitors/activators; includes enough active enzyme for entire plate
- Includes optimal substrate selected from a panel of succinylated sites
- 96-well plate included, but can be adapted to higher well format
- Control inhibitors included
- Suitable for high-throughput screening (Z’-factors >0.73)
- Optimal, specific SIRT5 substrate means low enzyme concentration, making ‘hit’ validation easy
The SIRT5 Fluorometric Drug Discovery Kit is a complete assay system designed to measure the lysyl desuccinylase activity of the recombinant human SIRT5 included in the kit. A black 96-well microplate is packaged with the kit, but it should be noted that reagents of the FLUOR DE LYS® system have also been successfully employed in other formats, including cuvettes and 384-well plates. The SIRT5 Fluorometric Activity Assay is based on the unique FLUOR DE LYS®-Succinyl Substrate/Developer combination. The assay procedure has two steps. The FLUOR DE LYS®-Succinyl Substrate, which comprises a single lysine residue, Nε-succinylated on its side-chain, is first incubated with human recombinant SIRT5 together with the cosubstrate NAD+. Desuccinylation of FLUOR DE LYS®-Succinyl sensitizes it so that, in the second step, treatment with the FLUOR DE LYS® Developer produces a fluorophore. Use of a succinylated, rather than acetylated substrate with SIRT5 results in readily observed saturation kinetics and a greater than 1000-fold increase in assay sensitivity.
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Performing the FLUOR DE LYS®-Succinyl SIRT5 Activity Assay. The procedure is done in two stages First, the components of the desuccinylation reaction (SIRT5, buffer or test compound, substrates) are mixed. Following an incubation in which substrate desuccinylation takes place, Developer is added and mixed. This stops the deacetylation and produces the fluorescent signal. The fluorescent signal develops and can be read in less than 15 min.

Dependence of SIRT5 Kinetics on the Concentration of NAD+. Initial desuccinylation rates of SIRT5 (12 Ū/10 ng) were determined with 20 min. incubations (37 °C) in the presence of 0.2 mM FLUOR DE LYS®-Succinyl and the indicated concentrations of NAD+. Reactions were stopped with FLUOR DE LYS® Developer/2 mM nicotinamide and fluorescence measured (Ex. 360 nm, Em. 460 nm). Each point represents the mean of three determinations and the error bars are standard deviations. The line is a non-linear least-squares fit to the Michaelis-Menten equation (Graphpad Prism). The Km for NAD+ was 390 µM and the Vmax was 1300 pmol/min./µg.



Product Details
Alternative Name |
Sirtuin 5 fluorescent assay kit |
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Application |
Activity assay, Cell-based assays, Fluorescent detection, HTS |
Contents |
BML-SE555-9090 SIRT5 (Sirtuin 5) (human, rec.) BML-KI105-0300 FLUOR DE LYS® Developer Concentrate (20x) |
Technical Info / Product Notes |
Most sirtuin enzymes, also known as class III histone deactylases (class III HDACs), catalyze a reaction which couples deacetylation of protein Ne-acetyllysine residues to the formation of O-acetyl-ADP-ribose and nicotinamide, from the oxidized form of nicotinamide adenine dinucleotide (NAD+). Some sirtuins, notably human SIRT4 and SIRT6, are reported to catalyze an alternative reaction, the transfer of an ADP-ribosyl group from NAD+ to proteins, although the physiological relevance of these reactions is in question. Sirtuin homologs are found in all forms of life, including the archaea, the bacteria and both unicellular and multicellular eukaryotes. The founding exemplar of the group, Sir2 from baker’s yeast (Saccharomyces cerevisiae), was named for its role in gene-silencing (Silent information regulator 2). Transcriptional silencing by Sir2 is linked to its deacetylation of lysines in the N-terminal tails of the histones in chromatin, hence the classification as a class III HDAC. Lysine deacetylation by sirtuins, however, extends beyond histones. Targets of sirtuin regulatory deacetylation include mammalian transcription factors such as p53, the cytoskeletal protein, tubulin, the bacterial enzyme, acetyl-CoA synthetase and its mammalian homologs. |
UniProt ID |
Q9NXA8 |
Handling & Storage
Use/Stability |
Store all components except the microplates and instruction booklet at -70°C. The SIRT5 enzyme, BML-SE555, must be handled with particular care in order to retain maximum enzymatic activity. Defrost it quickly in a RT water bath or by rubbing between fingers, then immediately store on an ice bath. The remaining, unused enzyme should be refrozen quickly, by placing at -70°C. If possible, snap freeze in liquid nitrogen or a dry ice/ethanol bath. To minimize the number of freeze/thaw cycles, aliquot into separate tubes and store at -70°C. The 20x Developer (BML-KI105) can be prone to precipitation if thawed too slowly. It is best to thaw this reagent in a room temperature water bath and, once thawed, transfer immediately onto ice. As with the SIRT5, it is best to refreeze unused portion in liquid nitrogen or a dry ice/ethanol bath. |
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Handling |
Avoid freeze/thaw cycles. |
Long Term Storage |
-80°C |
Shipping |
Dry Ice |
Regulatory Status |
RUO – Research Use Only |
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- Human SIRT5 variants with reduced stability and activity do not cause neuropathology in mice: Yuan, T., Kumar, S., et al.; iScience 27, 109991 (2024), Abstract
- SIRT5variants from patients with mitochondrial disease are associated with reduced SIRT5 stability and activity, but not with neuropathology: Yuan, T., Kumar, S., et al.; bioRxiv , (2023)
- Identification of the subtype-selective Sirt5 inhibitor balsalazide through systematic SAR analysis and rationalization via theoretical investigations: C. Glas, et al.; Eur. J. Med. Chem. 206, 112676 (2020), Application(s): Screening the inhibitory activity of synthesized compounds, Abstract
- Pharmacophore modeling and virtual screening studies to identify novel selective SIRT2 inhibitors: G. Eren, et al.; J. Mol. Graph. Model. 10, 1313 (2019), Abstract
- SIRT5 promotes IDH2 desuccinylation and G6PD deglutarylation to enhance cellular antioxidant defense: L. Zhou, et al.; EMBO Rep. 17, 811 (2016), Abstract — Full Text
- SIRT5 regulation of ammonia-induced autophagy and mitophagy: L. Polletta, et al.; Autophagy 11, 253 (2015), Abstract — Full Text
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