Recombinant Human Histone Deacetylase 8 Protein, CF Summary
Met1-Val377, with an N-terminal Met and 7-His tag
CF stands for Carrier Free (CF). We typically add Bovine Serum Albumin (BSA) as a carrier protein to our recombinant proteins. Adding a carrier protein enhances protein stability, increases shelf-life, and allows the recombinant protein to be stored at a more dilute concentration. The carrier free version does not contain BSA.
In general, we advise purchasing the recombinant protein with BSA for use in cell or tissue culture, or as an ELISA standard. In contrast, the carrier free protein is recommended for applications, in which the presence of BSA could interfere.
|Formulation||Supplied as a 0.2 μm filtered solution in Tris, NaCl and Glycerol.|
|Shipping||The product is shipped with polar packs. Upon receipt, store it immediately at the temperature recommended below.|
|Stability & Storage:||Use a manual defrost freezer and avoid repeated freeze-thaw cycles.
- 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
- Dilute rhHDAC8 to 0.02 µg/µL in Assay Buffer.
- Dilute Substrate to 500 µM in Assay Buffer.
- 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.
- Cover the plate with parafilm or a plate sealer and incubate at 37 °C for 1 hour.
- After incubation, add 50 µL of Stop Solution to all wells.
- Cover and incubate for 15 minutes at room temperature.
- For the Blank, add 50 µL stop solution, immediately followed by 25 µL of substrate solution and incubate for 15 minutes at room temperature.
- Read at excitation and emission wavelengths of 380 nm and 460 nm (top read), respectively, in endpoint mode.
- 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
Background: Histone Deacetylase 8/HDAC8
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).
- Hu, E. et al. (2000) J. Biol. Chem. 275:15254.
- Annemieke, J et al. (2003) Biochem. J. 370:737.
- Gray, S. and T. Ekstrom (2001) Exp. Cell Res. 262:75.
- Yang, X. and S. Gregoire (2005) Mol. Cel. Biol. 25:2873.
- Waltregny, D. et al. (2004) Am. J. Pathol. 165:553.
- De Levak, L. et al. (2006) Am. J. Surg. Pathol. 30:319.
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Fluorogenic Peptide Substrates
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