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HDAC2 (full-length) (human), (recombinant) (His-tag)

Highly active
 
BML-SE533-0050 50 µg 432.00 USD
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Product Specification

Alternative Name:Histone deacetylase 2
 
Sequence:HDAC2 from human cDNA. Sequence is identical to residues 1-582 at Genbank accession# NM_00157.2.
 
MW:~66 kDa
 
Source:Produced in insect cells.
 
UniProt ID:Q92769
 
Formulation:Liquid. In 50mM TRIS, pH 8.0, 138 mM NaCl and 10% glycerol.
 
Purity Detail:Partially purified by single-step affinity chromatography and gel filtration.
 
Specific Activity:≥0.5 U/µg. One U=1 pmol/min at 37°C, 100 µM, FLUOR DE LYS®-SIRT1 substrate (p53 K(Ac)-382 fluorogenic substrate, Prod. No. BML-KI177).
 
Shipping:Shipped on Dry Ice
 
Long Term Storage:-80°C
 
Use/Stability:The enzyme is stable on ice for the time typically required to set up an experiment (30-60 min.), but may lose activity with prolonged storage on ice. It is recommended that thawing and dilution of the enzyme be done within as short a time as possible before start of the assay. The remaining, unused, undiluted enzyme should be refrozen quickly by, for example, snap freezing in a dry/ice ethanol bath or liquid nitrogen. Freezing and storage of diluted enzyme is not recommended.
 
Scientific Background:HDAC2, like the highly homologous HDAC1, is a class I HDAC first identified as a human homolog of the yeast histone deacetylase Rpd3. HDAC2 found, along with HDAC1 in the Sin3, NuRD. In contrast to class II HDACs, which repress cardiac hypertrophy, HDAC2 activity is required for the cardiac hypertrophic response.
 
Protocol:Assay of Full-length HDAC2 (Prod. No. BML-SE533) with FLUOR DE LYS®-SIRT1 substrate (Prod. No. BML-KI177) & FLUOR DE LYS® Developer II (Prod. No. BML-KI176)
Components of Assay
HDAC Assay Buffer II (Prod. No. BML-KI422): (50 mM Tris/Cl, pH 8.0, 137mM NaCl, 2.7mM KCl, 1mM MgCl2, 1mg/ml BSA (fatty acid free BSA, e.g. Sigma catalog # BML-A-3803))
HDAC2 (Prod. No. BML-SE533): Dilute HDAC2 to 100ng/μl with HDAC Assay Buffer II (Prod. No. BML-KI422) to provide 5 μl per well. Keep on ice until use.
FLUOR DE LYS®-SIRT1 substrate (BML-KI177)*: Thaw quickly and keep on ice. Dilute 5mM stock in HDAC Assay Buffer II.
2x Substrate Solution: Prepare a 2x substrate solution by diluting the 5 mM FLUOR DE LYS®-SIRT1 substrate (Prod. No. BML-KI177) in HDAC Assay Buffer II (Prod. No. BML-KI422). Each assay well will require 25μl (see below). For example, prepare 1ml of 40μM substrate (for final 20μM) by mixing 8μl of 5mM FLUOR DE LYS®-SIRT1 substrate (Prod. No. BML-KI177) and 992μl HDAC Assay Buffer II (Prod. No. BML-KI422). Warm to 37°C before use. (NOTE: HDAC2’s Km for FLUOR DE LYS®-SIRT1 substrate (Prod. No. BML-KI177) is ~19μM. Therefore, a reasonable substrate concentration for inhibitor screening would be 20μM FLUOR DE LYS®-SIRT1 substrate (Prod. No. BML-KI177), while 500μM would be more suitable for a specific activity measurement at a saturating substrate concentration.)
Trichostatin A (Prod. No. BML-GR309; HDAC Inhibitor): Prepare a 0.2mM stock in dimethylsulfoxide (DMSO). DMSO stock may be stored at -20°C. The 0.2mM stock will be diluted 100-fold in 1x Developer II in order to stop HDAC activity at the start of the signal development process. To prepare stocks for use in testing trichostatin inhibition (i.e. for addition to the deacetylation phase of the reaction), dilute 0.2mM stock to 10μM in HDAC Assay Buffer II (e.g. 5μl plus 95μl) and make further serial dilutions in buffer to 1μM and 0.1μM. Addition of 2.5μl of the 10μM stock per well will result in strong inhibition (final [trichostatin A] = 500nM), while addition of 2.5μl of the 0.1μM stock per well will produce ~50% inhibition with 5μM FLUOR DE LYS®-SIRT1 substrate (Prod. No. BML-KI177) as substrate (final [trichostatin A] = 5nM).
FLUOR DE LYS® Developer II (5x Concentrate, Prod. No. BML-KI176): Shortly before use, dilute 5x stock solution to 1x plus 2μM trichostatin A. For example, prepare 1ml by mixing 200μl of the 5x Concentrate, 790μl HDAC Assay Buffer II (Prod. No. BML-KI422) and 10μl 0.2mM trichostatin A in DMSO. Store the 1x Developer II plus trichostatin on ice until use. Do not store excess, but prepare freshly as needed.
FLUOR DE LYS® Deacetylated Standard (Prod. No. BML-KI142): Dilute the 10mM stock in DMSO to 1μM with HDAC Assay Buffer II (Prod. No. BML-KI422).
½ Volume 96-well white micro-plate (Prod. No. BML-KI110)

Reaction Condition Examples
  1. Designate wells for four reactions: 30 min rxn; 30 min rxn plus trichostatin A; 0 min rxn and a Standard well.
  2. Add 20μl of HDAC Assay Buffer II to the 30 min rxn well and the 0 min rxn well. To the third well (30 min. plus trichostatin) add 2.5μl of 10μM or 0.1μM trichostatin A plus 17.5μl of HDAC Assay Buffer II. Allow to equilibrate to assay temperature (37°C). (Leave Standard well empty until step 7).
  3. Add 5μl of diluted HDAC2 (Prod. No. BML-SE533, 100ng/μl) to the wells for the 0 min, 30 min, and 30 min. plus trichostatin rxns.
  4. To start reactions, add 25μl of the 2x Substrate (37°C) to both 30 min reaction wells. Allow reactions to run 30 min @ 37°C.
  5. Add 50μl of 1x Developer II plus trichostatin A to both 30 min. reaction wells.
  6. To the 0 min rxn well, add 50μl of 1x Developer II plus trichostatin A, immediately followed by 25μl of 2x substrate.
  7. For the standard, mix 50μl of 1μM standard with 50μl of 1x Developer II plus trichostatin A in the fourth well.
  8. Allow 45 min. at 37°C for signal to develop and then read plate in a microplate-reading fluorimeter capable of excitation at a wavelength in the range of 350-380 and detection of emitted light in the range of 440-460 nm.
  9. Data analysis: Determine the ΔAFU (Arbitrary Fluorescence Units) for the two 30 min rxns. (AFU of 30 min rxn. (with or without trichostatin) minus AFU of the 0 min rxn). Determine AFU/pmol by dividing the Deacetylated standard reading (AFU) by 50pmol. Calculate pmol of substrate deacetylated in 30 min (divide ΔAFU by AFU/pmol).
 

General Literature References

Hdac2 regulates the cardiac hypertrophic response by modulating Gsk3 beta activity: C.M. Trivedi, et al.; Nat. Med. 13, 324 (2007), Abstract;
Histone deacetylase 2-mediated deacetylation of the glucocorticoid receptor enables NF-kappaB suppression: K. Ito, et al.; J. Exp. Med. 203, 7 (2006), Abstract;
Histone deacetylases (HDACs): characterization of the classical HDAC family: A.J. de Ruijter, et al.; Biochem. J. 370, 737 (2003), Abstract;
CoREST is an integral component of the CoREST- human histone deacetylase complex: A. You, et al.; PNAS U.S.A. 98, 1454 (2001), Abstract;
Regulation of transcription factor YY1 by acetylation and deacetylation: Y.L. Yao, et al.; Mol. Cell Biol. 21, 5979 (2001), Abstract;
Analysis of the NuRD subunits reveals a histone deacetylase core complex and a connection with DNA methylation.: Y. Zhang et al.; Genes Dev. 13, 1924 (1999), Abstract;
Histone deacetylases and SAP18, a novel polypeptide, are components of a human Sin3 complex: Y. Zhang, et al.; Cell 89, 357 (1997), Abstract;
Histone deacetylases associated with the mSin3 corepressor mediate mad transcriptional repression: C.D. Laherty, et al.; Cell 89, 349 (1997), Abstract;
Transcriptional repression by YY1 is mediated by interaction with a mammalian homolog of the yeast global regulator RPD3: W.M. Yang, et al.; PNAS U.S.A. 93, 12845 (1996), Abstract;

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