Recombinant Human Histone Deacetylase 8 Protein, CF

• Assay Buffer: 15 mM Tris, 0.25 mM EDTA, 250 mM NaCl, 0.1% (w/v) PEG 8000, pH 8.0
• Stop Solution: 8 ng/μL Recombinant Human Active Trypsin 3/PRSS3 (Catalog # 3714-SE), 2 μM Trichostatin A (Sigma, Catalog # T8552), 50 mM Tris, 100 mM NaCl, 30% (v/v) Isopropanol, pH 8.0
• Recombinant Human Histone Deacetylase 8/HDAC8 (rhHDAC8) (Catalog # 4359-DA)
• Substrate: Ac-Arg-Gly-Lys(Ac)-AMC (Catalog # ES016)
• F16 Black Maxisorp Plate (Nunc, Catalog # 475515)
• Fluorescent Plate Reader (Model: SpectraMax Gemini EM by Molecular Devices) or equivalent

1. Dilute rhHDAC8 to 0.02 µg/µL in Assay Buffer.
2. Dilute Substrate to 500 µM in Assay Buffer.
3. Combine 25 µL of 0.02 µg/µL rhHDAC8 and 25 µL of Substrate in a plate. Add 25 µL of 0.02 µg/µL rhHDAC8 alone as a Blank.
4. Cover the plate with parafilm or a plate sealer and incubate at 37 °C for 1 hour.
5. After incubation, add 50 µL of Stop Solution to all wells.
6. Cover and incubate for 15 minutes at room temperature.
7. For the Blank, add 50 µL stop solution, immediately followed by 25 µL of substrate solution and incubate for 15 minutes at room temperature.
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 Substrate Blank

     **Derived using calibration standard 7-amino, 4-Methyl Coumarin (Sigma, Catalog # A-9891).

Per Well:

• rhHDAC8: 0.5 µg
• Substrate: 125 µM