Recombinant Human Sirtuin 5/SIRT5 His-tag Protein, CF

• Assay Buffer: 25 mM Tris, 150 mM NaCl, 1 mM DTT, pH 8.0
• Stop Solution: 8 ng/µL Recombinant Human Active Trypsin 3/PRSS3 (Catalog # 3714-SE), 4 mM Nicotinamide (Sigma, Catalog # 72340), 50 mM Tris, 100 mM NaCl, 30% (v/v) isopropanol, pH 8.0
• Recombinant Human Sirtuin 5/SIRT5 (rhSIRT5) (Catalog # 10270-DA)
• Nicotinamide adenine dinucleotide sodium salt ( beta -NAD) (Sigma, Catalog # N6522), 100 mM stock in diH2O
• Substrate: FLUOR DE LYS®-Succinyl, Desuccinylase Substrate (5 mM) (Enzo Life Sciences Int., Catalog # BML-KI590)
• F16 Black Maxisorp Plate (Nunc, Catalog # 475515)
• Fluorescent Plate Reader (Model: SpectraMax Gemini EM by Molecular Devices) or equivalent

1. Dilute rhSIRT5 to 1 ng/µL in Assay Buffer.
2. Dilute Substrate to 100 µM in Assay Buffer containing 2 mM beta -NAD.
3. Combine 25 µL of 1 ng/µL rhSIRT5 and 25 µL of dilute Substrate in plate. For Control, load 25 µL of 1 ng/µL rhSIRT5 alone.
4. Seal plate and incubate at 37 °C for 30 minutes.
5. Add 50 µL of Stop Solution to all wells and mix.  For Control well(s), add 25 µL of dilute Substrate after addition of Stop Solution.
6. Seal plate and incubate at room temperature for 15 minutes.
7. Read at excitation and emission wavelengths of 380 nm and 460 nm (top read), respectively, in endpoint mode.
8. Calculate specific activity:

*Adjusted for Control
**Derived using calibration standard 7-amino, 4-Methyl Coumarin (AMC) (Sigma, Catalog # A9891).

• rhSIRT5: 0.025 µg
• Substrate: 25 µM
• beta -NAD: 0.5 mM