Recombinant Human Sirtuin 3/SIRT3 Protein, CF

• 6 months from date of receipt, -20 to -70 °C as supplied.
• 3 months, -20 to -70 °C under sterile conditions after opening.

• Assay Buffer: 25 mM Tris, 150 mM NaCl, 1 mM DTT, pH 9.0
• Stop Buffer: 50 mM Tris, 100 mM NaCl, 30% (v/v) Isopropanol, pH 8.0
• Recombinant Human Sirtuin 3/SIRT3 (rhSIRT3) (Catalog # 7488-DA)
• Fluorogenic Peptide Substrate Ac-Arg-Gly-Lys(Ac)-AMC  (Catalog # ES016), 20 mM in DMSO
• beta -nicotinamide adenine dinucleotide hydrate ( beta -NAD) (Sigma, Catalog # N6522, 100 mM in diH2O)
• Recombinant Mouse Active Trypsin 3/PRSS3 (Catalog # 7235-SE)
• Nicotinamide (Sigma, Catalog # 72340, 100 mM in diH2O)
• F16 Black Maxisorp Plate (Nunc, Catalog # 475515)
• Fluorescent Plate Reader (Model: SpectraMax Gemini EM by Molecular Devices) or equivalent

1. Dilute rhSIRT3 to 40 ng/µL in Assay Buffer.
2. Dilute Substrate to 1 mM in Assay Buffer containing 2 mM beta -NAD.
3. Load into plate 25 µL of 40 ng/µL rhSIRT3 in triplicate and start the reaction by adding 25 µL of 1 mM Substrate. As a control, load 25 µL of 40 ng/µL rhSIRT3 in triplicate.
4. Seal plate and incubate at 37 °C for 30 minutes.
5. Prepare Stop Solution by combining Recombinant Mouse Trypsin 3/PRSS3 and Nicotinamide in Stop Buffer to final concentrations of 0.2 ng/µL and 4 mM, respectively.
6. Add 50 µL of Stop Solution to all wells. To the control wells, add 25 µL of 1 mM Substrate mixture after addition of Stop Solution.
7. Seal plate tightly with plate sealer and incubate at room temperature for 15 minutes.
8. Read at excitation and emission wavelengths of 380 nm and 460 nm (top read), respectively, in endpoint mode.
9. Calculate specific activity:

     *Adjusted for Control
     **Derived using calibration standard 7-amino, 4-Methyl Coumarin (AMC) (Sigma, Catalog # A9801).

• rhSIRT3: 1.0 µg
• Substrate: 250 µM