The remaining, unused enzyme should be refrozen quickly, by placing at -70☌. Defrost it quickly in a RT water bath or by rubbing between fingers, then immediately store on an ice bath. The SIRT5 enzyme, BML-SE555, must be handled with particular care in order to retain maximum enzymatic activity. Store all components except the microplates and instruction booklet at -70☌. 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.

Desuccinylation of FLUOR DE LYS ® -Succinyl sensitizes it so that, in the second step, treatment with the FLUOR DE LYS ® Developer produces a fluorophore. 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+. The SIRT5 Fluorometric Activity Assay is based on the unique FLUOR DE LYS ® -Succinyl Substrate/Developer combination. 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 Drug Discovery Kit is a complete assay system designed to measure the lysyl desuccinylase activity of the recombinant human SIRT5 included in the kit.

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