Recombinant Human Histone Deacetylase 8 Protein, CF

• Reactivity: Hu
• Applications: Enzyme Activity
• Format: Carrier-Free

Recombinant Human Histone Deacetylase 8 Protein, CF Summary

• Details of Functionality: Measured by its ability to remove the acetyl group from a fluorogenic peptide substrate Ac-RGK(Ac)-AMC (Catalog # ES016) in a coupled assay. The specific activity is >1.5 pmol/min/µg, as measured under the described conditions.
• Source: Spodoptera frugiperda, Sf 21 (baculovirus)-derived human Histone Deacetylase 8/HDAC8 protein
  Met1-Val377, with an N-terminal Met and 7-His tag
• Accession #: AAH50433
• N-terminal Sequence: No results obtained: Met predicted
• Protein/Peptide Type: Recombinant Enzymes
• Gene: HDAC8
• Purity: >85%, by SDS-PAGE under reducing conditions and visualized by silver stain
• Endotoxin Note: <1.0 EU per 1 μg of the protein by the LAL method.

Applications/Dilutions

• Dilutions:
  • Enzyme Activity
• Theoretical MW: 43 kDa.
  Disclaimer note: The observed molecular weight of the protein may vary from the listed predicted molecular weight due to post translational modifications, post translation cleavages, relative charges, and other experimental factors.
• SDS-PAGE: 48 kDa, reducing conditions

Packaging, Storage & Formulations

• Storage: Use a manual defrost freezer and avoid repeated freeze-thaw cycles.
  • 6 months from date of receipt, -20 to -70 °C as supplied.
  • 3 months, -20 to -70 °C under sterile conditions after opening.
• Buffer: Supplied as a 0.2 μm filtered solution in Tris, NaCl and Glycerol.
• Purity: >85%, by SDS-PAGE under reducing conditions and visualized by silver stain
• Assay Procedure:
  • 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:

  Specific Activity (pmol/min/µg) = (Adjusted Fluorescence* (RFU) x Conversion Factor** (pmol/RFU)) / (Incubation time (min) x amount of enzyme (µg))

  *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

Notes

This product is produced by and ships from R&D Systems, Inc., a Bio-Techne brand.

Alternate Names for Recombinant Human Histone Deacetylase 8 Protein, CF

• EC 3.5.1.98
• HD8
• HDAC8
• HDACL1
• Histone Deacetylase 8
• histone deacetylase-like 1
• RPD3

Background

Human Histone Deacetylase 8 (HDAC8) is a member of the class I Histone Deacetylases (HDACs) (1, 2). HDACs are important enzymes for the transcriptional regulation of gene expression in eukaryotic cells (3). HDACs catalyze the removal of acetyl groups from lysines near the N-termini of histones. Human HDACs have been implicated in a variety of human diseases such as cardiomyopathy, osteodystrophy, neurodegenerative disorders, aging and cancer (4). Expression of HDAC8 is restricted to cells showing smooth muscle differentiation in normal human tissue and is a novel marker of smooth muscle differentiation (5, 6). Like other class I and II HDAC members, the activity of HDAC8 is sensitive to HDAC inhibitor trichostatin A (1).

1. Hu, E. et al. (2000) J. Biol. Chem. 275:15254.
2. Annemieke, J et al. (2003) Biochem. J. 370:737.
3. Gray, S. and T. Ekstrom (2001) Exp. Cell Res. 262:75.
4. Yang, X. and S. Gregoire (2005) Mol. Cel. Biol. 25:2873.
5. Waltregny, D. et al. (2004) Am. J. Pathol. 165:553.
6. De Levak, L. et al. (2006) Am. J. Surg. Pathol. 30:319.
   
   

Bioinformatics

• Gene Symbol: HDAC8
• Uniprot:
  • AAH50433()