HDAC Activity Colorimetric Assay Kit

Details for Product No. ABIN411893
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Target Name (Antigen)
Synonyms
ARABIDOPSIS HISTONE DEACETYLASE 1, ARABIDOPSIS HISTONE DEACETYLASE 19, ATHD1, ATHDA19, ATRPD3A, F20D10.250, F20D10_250, HDA1, HDA19, HISTONE DEACETYLASE, HISTONE DEACETYLASE 19, HISTONE DEACETYLASE19, ... show more
ARABIDOPSIS HISTONE DEACETYLASE 1, ARABIDOPSIS HISTONE DEACETYLASE 19, ATHD1, ATHDA19, ATRPD3A, F20D10.250, F20D10_250, HDA1, HDA19, HISTONE DEACETYLASE, HISTONE DEACETYLASE 19, HISTONE DEACETYLASE19, RPD3A, histone deacetylase 1, ARABIDOPSIS HISTONE DEACETYLASE 2, ATHD2, ATHD2B, HD2, HDA4, HDT02, HDT2, HISTONE DEACETYLASE 2, MDJ22.7, MDJ22_7, histone deacetylase 2B, Tb05.26K5.290 show less
Reactivity
Mammalian
(1), (1), (1), (1), (1), (1), (4), (2), (1), (4), (1), (1), (5), (1)
Application
Activity Assay (AcA)
Pubmed 21 references available
Quantity 100 tests
Options
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Purpose Inhibition of histone deacetylases (HDACs) has been implicated to modulate transcription and to induce apoptosis or differentiation in cancer cells. However, screening HDAC inhibitory compounds has proven to be difficult over the past due to the lack of convenient tools for analyzing HDAC activity. The new Colorimetric HDAC Activity Assay Kit provides a fast and convenient colorimetric method that eliminates radioactivity, extractions, or chromatography, as used in the traditional assays. The new method requires only two easy steps, both performed on the same microtiter plate. First, the HDAC colorimetric substrate, which comprises an acetylated lysine side chain, is incubated with a sample containing HDAC activity (e.g., HeLa nuclear extract or your own samples). Deacetylation of the substrate sensitizes the substrate, so that, in the second step, treatment with the Lysine Developer produces a chromophore. The chromophore can be easily analyzed using an ELISA plate reader or spectrophotometer. The assay is well suited for high throughput screening applications. HDAC inhibitors and antibodies are also available separately.
Detection Method Colorimetric
Specificity The new Colorimetric HDAC Activity Assay Kit provides a fast and convenient colorimetric method that eliminates radioactivity, extractions, or chromatography, as used in the traditional assays. The new method requires only two easy steps, both performed on the same microtiter plate. first, the HDAC colorimetric substrate, which comprises an acetylated lysine side chain, is incubated with a sample containing HDAC activity (e.g., HeLa nuclear extract or your own samples). Deacetylation of the substrate sensitizes the substrate, so that, in the second step, treatment with the Lysine Developer produces a chromophore. The chromophore can be easily analyzed using an ELISA plate reader or spectrophotometer. The assay is well suited for high throughput screening applications. HDAC inhibitors and antibodies are also available separately
Characteristics Colorimetric HDAC Activity Assay Kit: Measure Histone Deacetylase (HDAC) Activity in Just Two Easy Steps. Convenient, High-Throughput Adaptable Colorimetric Assay.
Components HDAC Substrate [Ac-Lys(Ac)-pNA, 10 mM]
10X HDAC Assay Buffer
Lysine Developer
HDAC Inhibitor (Trichostatin A, 1 mM)
HeLa Nuclear Extract (5 mg/ml)
Deacetylated Standard (Ac-Lys-pNA, 10 mM)
Alternative Name HDAC (HDAC ELISA Kit Abstract)
Background Inhibition of histone deacetylases (HDACs) has been implicated to modulate transcription and to induce apoptosis or differentiation in cancer cells. However, screening HDAC inhibitory compounds has proven to be difficult over the past due to the lack of convenient tools for analyzing HDAC activity.
Application Notes Well suited for high throughput applications. HDAC inhibitors and substrates and related antibodies are also available separately.
Comment

Absorbance (400 or 405 nm)
Simple two-step procedure, takes around than 1 hour
Fast and convenient
The assay method eliminates radioactivity/extractions/ and/or chromatography as used in the traditional assays.

Assay Time 1 h
Protocol A. General Consideration: Read the entire protocol before beginning the procedure. The HeLa nuclear extract and Lysine Developer should be refreeze immediately at -20 or -70 °C after each use to avoid loss of activity. If positive and negative controls are designed, the kit provides sufficient reagents for 5 positive control assays with the HeLa Nuclear Extract and 5 Negative Control assays with the HDAC Inhibitor, Trichostatin A. Using 96-well plates with U-shape bottom. Flat bottom may give a little low value. B.
1. Dilute test samples (50-200 µg of nuclear extract or cell lysate) to 85 µL (final volume) of ddH 2 0 in each well (For background reading, add 85 µL ddH 2 0 only). For positive control, dilute 10 µL of HeLa nuclear extract with 75 µL ddH 2 0. For negative control, dilute yoursample into 83 µL of ddH 2 0 and then add 2 µL of Trichostatin, or use a known sample containing no HDAC activity.
2. Add 10 µL of the 10X HDAC Assay Buffer to each well.
3. Add 5 µL of the HDAC colorimetric substrate to each well. Mix thoroughly.
4. Incubate plates at 37 °C for 1 hour (or longer if desired).
5. Stop the reaction by adding 10 µL of Lysine Developer and mix well. Incubate the plate at 37 °C for 30 min.
6. Read sample in an ELISA plate reader at 400 or 405 nm. Signal is stable for several hours at room temperature. HDAC activity can be expressed as the relative O.D. value per µg protein sample. C. Standard Curve (optional):
1. If desired, a standard curve can be prepared using the known amount of the Deacetylated Standard included in the kit. The exact concentration range of the Deacetylase Standard will vary depending on the each individual plate reader and the exact wavelength used. We recommend starting with a dilution range of 10-100 µM in Assay Buffer.
2. Add 90 µL each of the dilutions and also 10 µL of the 10X Assay Buffer into a set of wells on the microtiter plate. Use 90 µL of H2O and 10 µL of 10X Assay Buffer as zero
3. Add 10 µL of Lysine Developer to each well and incubate at 37 °C for 30 min (Note: Incubation time should be kept the same for both standard and test samples.)
4. Read samples in an ELISA plate reader at 400 or 405 nm.
5. Plot O.D. value (y-axis) versus concentration of the Deacetylated Standard (x-axis). Determine the slope as delta O.D./ µM.
6. Based on the slope, you can determine the absolute amount of deacetylated lysine generated in your sample.
Restrictions For Research Use only
Storage -20 °C
Expiry Date 12 months
Product cited in: Yang, Li, Chen et al.: "PARP-1 mediates LPS-induced HMGB1 release by macrophages through regulation of HMGB1 acetylation." in: Journal of immunology (Baltimore, Md. : 1950), Vol. 193, Issue 12, pp. 6114-23, 2014 (PubMed).

Zhang, DeNicola, Qin et al.: "Specific inhibition of HDAC4 in cardiac progenitor cells enhances myocardial repairs." in: American journal of physiology. Cell physiology, Vol. 307, Issue 4, pp. C358-72, 2014 (PubMed).

Wu, Zheng, Huang et al.: "Lithium down-regulates histone deacetylase 1 (HDAC1) and induces degradation of mutant huntingtin." in: The Journal of biological chemistry, Vol. 288, Issue 49, pp. 35500-10, 2013 (PubMed).

Spallotta, Cencioni, Straino et al.: "A nitric oxide-dependent cross-talk between class I and III histone deacetylases accelerates skin repair." in: The Journal of biological chemistry, Vol. 288, Issue 16, pp. 11004-12, 2013 (PubMed).

Alsadany, Shehata, Mohamad et al.: "Histone deacetylases enzyme, copper, and IL-8 levels in patients with Alzheimer's disease." in: American journal of Alzheimer's disease and other dementias, Vol. 28, Issue 1, pp. 54-61, 2013 (PubMed).

Gnana-Prakasam, Veeranan-Karmegam, Coothankandaswamy et al.: "Loss of Hfe leads to progression of tumor phenotype in primary retinal pigment epithelial cells." in: Investigative ophthalmology & visual science, Vol. 54, Issue 1, pp. 63-71, 2013 (PubMed).

Zhang, Qin, Zhao et al.: "Inhibition of histone deacetylases preserves myocardial performance and prevents cardiac remodeling through stimulation of endogenous angiomyogenesis." in: The Journal of pharmacology and experimental therapeutics, Vol. 341, Issue 1, pp. 285-93, 2012 (PubMed).

Minetti, Feige, Rosenstiel et al.: "Gαi2 signaling promotes skeletal muscle hypertrophy, myoblast differentiation, and muscle regeneration." in: Science signaling, Vol. 4, Issue 201, pp. ra80, 2011 (PubMed).

Singh, Thangaraju, Prasad et al.: "Blockade of dendritic cell development by bacterial fermentation products butyrate and propionate through a transporter (Slc5a8)-dependent inhibition of histone deacetylases." in: The Journal of biological chemistry, Vol. 285, Issue 36, pp. 27601-8, 2010 (PubMed).

Roccaro, Sacco, Jia et al.: "microRNA-dependent modulation of histone acetylation in Waldenstrom macroglobulinemia." in: Blood, Vol. 116, Issue 9, pp. 1506-14, 2010 (PubMed).

Zhou, Deng, Norseen et al.: "Regulation of Epstein-Barr virus origin of plasmid replication (OriP) by the S-phase checkpoint kinase Chk2." in: Journal of virology, Vol. 84, Issue 10, pp. 4979-87, 2010 (PubMed).

Liu, Mai, Sun: "Lysine acetylation regulates Bruton's tyrosine kinase in B cell activation." in: Journal of immunology (Baltimore, Md. : 1950), Vol. 184, Issue 1, pp. 244-54, 2009 (PubMed).

Farhana, Dawson, Dannenberg et al.: "SHP and Sin3A expression are essential for adamantyl-substituted retinoid-related molecule-mediated nuclear factor-kappaB activation, c-Fos/c-Jun expression, and cellular apoptosis." in: Molecular cancer therapeutics, Vol. 8, Issue 6, pp. 1625-35, 2009 (PubMed).

Bourguignon, Xia, Wong et al.: "Hyaluronan-mediated CD44 interaction with p300 and SIRT1 regulates beta-catenin signaling and NFkappaB-specific transcription activity leading to MDR1 and Bcl-xL gene expression and chemoresistance ..." in: The Journal of biological chemistry, Vol. 284, Issue 5, pp. 2657-71, 2009 (PubMed).

Tavera-Mendoza, Quach, Dabbas et al.: "Incorporation of histone deacetylase inhibition into the structure of a nuclear receptor agonist." in: Proceedings of the National Academy of Sciences of the United States of America, Vol. 105, Issue 24, pp. 8250-5, 2008 (PubMed).

Yao, Nyomba: "Hepatic insulin resistance induced by prenatal alcohol exposure is associated with reduced PTEN and TRB3 acetylation in adult rat offspring." in: American journal of physiology. Regulatory, integrative and comparative physiology, Vol. 294, Issue 6, pp. R1797-806, 2008 (PubMed).

Pandey, Ugale, Zhang et al.: "Brain chromatin remodeling: a novel mechanism of alcoholism." in: The Journal of neuroscience : the official journal of the Society for Neuroscience, Vol. 28, Issue 14, pp. 3729-37, 2008 (PubMed).

Zhao, Cheng, Zhang et al.: "Inhibition of histone deacetylases triggers pharmacologic preconditioning effects against myocardial ischemic injury." in: Cardiovascular research, Vol. 76, Issue 3, pp. 473-81, 2007 (PubMed).

Thangaraju, Gopal, Martin et al.: "SLC5A8 triggers tumor cell apoptosis through pyruvate-dependent inhibition of histone deacetylases." in: Cancer research, Vol. 66, Issue 24, pp. 11560-4, 2006 (PubMed).

Zeng, Xiao, Margariti et al.: "HDAC3 is crucial in shear- and VEGF-induced stem cell differentiation toward endothelial cells." in: The Journal of cell biology, Vol. 174, Issue 7, pp. 1059-69, 2006 (PubMed).

Pong, Roark, Ou et al.: "Mechanism of increased coxsackie and adenovirus receptor gene expression and adenovirus uptake by phytoestrogen and histone deacetylase inhibitor in human bladder cancer cells and the potential ..." in: Cancer research, Vol. 66, Issue 17, pp. 8822-8, 2006 (PubMed).

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Catalog No. ABIN411893
385.00 $
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Quantity
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100 tests
385.00 $

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