Recombinant Human Sirtuin 1/SIRT1 Protein, CF

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

• Assay Buffer: 50 mM Tris, 1 mM DTT, pH 9.0
• Stop Buffer: 50 mM Tris, 100 mM NaCl, 30% (v/v) isopropanol, pH 8.0
• Recombinant Human Sirtuin 1/SIRT1 (rhSIRT1) (Catalog # 7714-DA)
• Fluorogenic Peptide Substrate Ac-Arg-Gly-Lys(Ac)-AMC (Catalog # ES016)
• beta -nicotinamide adenine dinucleotide hydrate ( beta -NAD) (Sigma, Catalog # N6522), 100 mM in diH₂O
• Recombinant Mouse Active Trypsin 3/PRSS3 (Trypsin 3) (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 rhSIRT1 to 10 µg/mL in Assay Buffer.
2. Dilute Substrate to 500 µM in Assay Buffer containing 2 mM beta -NAD.
3. Load 25 µL of 10 µg/mL rhSIRT1 into the plate in triplicate and start the reaction by adding 25 µL of 500 µM Substrate. Include a control containing 25 µL of 10 ng/µL rhSIRT1 only.
4. Incubate sealed plate at 37 °C for 30 minutes.
5. Prepare Stop Solution by combining Trypsin 3 and Nicotinamide in Stop Buffer to final concentrations of 0.2 µg/mL and 4 mM, respectively.
6. Add 50 µL of Stop Solution to all wells. Add 25 µL of 500 µM Substrate to the enzyme control wells after the 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.

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

• rhSIRT1: 0.25 µg
• Substrate: 125 µM