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Epigenase HDAC Activity/Inhibition Direct Assay Kit (Colorimetric)


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Suggested Workflow
Nuclear Protein Extraction
HDAC Activity/Inhibition Assay
Schematic procedure of the Epigenase™ HDAC Activity/Inhibition Direct Assay Kit (Colorimetric).
Demonstration of high sensitivity of an HDAC activity assay achieved by using recombinant HDAC3 with the Epigenase™ HDAC Activity/Inhibition Direct Assay Kit (Colorimetric).
Demonstration of the inhibitory effect of an HDAC inhibitor detected by the Epigenase™ HDAC Activity/Inhibition Direct Assay Kit (Colorimetric). HDAC3 concentration: 20 ng/well.
Input Type: Nuclear Extracts, Purified Enzyme
Research Area: Histone Acetylation
Target Application: Activity Measurement
Vessel Format: 96-Well Plate
100% Guarantee: 6 months
Catalog No.SizePriceQty
P-4034-4848 assays $205.00 
P-4034-9696 assays $379.00 
Order now & get it by Thursday, October 24th  
Product Overview

The Epigenase™ HDAC Activity/Inhibition Direct Assay Kit is a complete set of optimized buffers and reagents for measuring the activity or inhibition of total HDAC enzymes in nuclear extracts or purified HDAC isoforms (HDACs 1-11) from a broad range of species such as mammals, plants, fungi, and bacteria, and in a variety of forms including, but not limited to, cultured cells and fresh and frozen tissues. The Epigenase™ HDAC Activity/Inhibition Direct Assay Kit is a further refinement of its predecessor, the EpiQuik™ HDAC Activity/Inhibition Assay Kit, a popular assay method for HDAC activity and inhibition. This latest method retains the simplicity, rapidness, high throughput capability, and non-radioactivity featured in the previous version, while offering the following advantages:

  • Improved kit composition enables background signals to be very low, which allows the assay to be more accurate, sensitive, reliable, and consistent.
  • Innovative colorimetric assay measures HDAC activity/inhibition by directly detecting HDAC-converted deacetylated products, rather than trypsin-based peptide cleavage, thus eliminating assay interference caused by DMSO, thiol-containing chemicals, trypsin, and cellular lysyl endopeptidases.
  • Both cell/tissue extracts and purified HDAC enzyme can be used, which allows for the detection of inhibitory effects of HDAC inhibitor in vivo andin vitro.
  • Novel assay principle allows high sensitivity to be achieved. The activity can be detected from as low as 0.5 ng of purified HDAC enzyme, which is about 10 fold higher than that obtained by trypsin-based peptide cleavage assays.
  • A deacetylated histone standard is included, which allows the specific activity of HDACs to be quantified.
  • Trichostatin A (TSA,) a potent inhibitor of class I and class II HDAC activity, is included as the positive control for the HDAC inhibition.
  • Strip-well microplate format makes the assay flexible and quick: manual or high throughput analysis can be completed within 3.5 hours.

Back ground Information
Acetylation of epsilon amino group of specific lysine residue contained in core histones is one of the most robust epigenetic marks and is essential for the regulation of multiple cellular processes. The acetylation of histone by histone acetyltransferases (HAT) seems to be of particular significance, as it is associated with active regions of the genome. In contrast, histone deacetylation by histone deacetylases (HDAC) leads to transcription repression. So far, at least 4 classes of HDACs have been identified. Class I HDACs include 1, 2, 3 and 8. Class II HDACs are comprised of 4, 5, 6, 7, 9 and 10. Class III enzymes, known as the sirtuins, require NAD+ cofactors and include SIRTs 1 through 7. Class IV enzymes, which contains only HDAC11, have features of both Class I and II.

HDACs are tightly involved in cell cycle regulation, cell proliferation, and development of human diseases including cancer, cardiovascular and pulmonary diseases, and neurological diseases. For example, in various tumor cells, HDAC enzymes are found to be overexpressed. In chronic obstructive pulmonary disease (COPD) HDACs are observed to be decreased. In Huntington’s disease, HDAC4 is critically involved. Detection of activity and inhibition of HDACs would be important in elucidating mechanisms of epigenetic regulation of gene activation and silencing and would benefit cancer diagnostics and therapeutics.

Principle & Procedure
The Epigenase™ HDAC Activity/Inhibition Assay Kit contains all reagents necessary for the measurement of HDAC activity/inhibition. In this assay, an acetylated histone HDAC substrate is stably coated onto the microplate wells. Active HDACs bind to the substrate and removes acetyl groups from the substrate. The HDAC-deacetylated products can be recognized with a specific antibody. The ratio or amount of deacetylated products, which is proportional to the enzyme activity, can then be colorimetrically measured by reading the absorbance in a microplate spectrophotometer at 450 nm. The activity of the HDAC enzyme is proportional to the OD intensity measured. 

  Epigenase™ Kit Other Supplier's Kit
Assay Principle Immunoaffinity-based end product direct measurement Endopeptidase cleavage-based indirect detection
Assay Format 96-Well Microplate-based 96-Well Microplate-based
Sensitivity Excellent: detect from as low as 0.5 ng of enzyme Poor: detect from 5 ng enzyme
Signal-Noise Ratio >20 with very low background <5 with high background
Interference by Chemical Reagents No Yes, affected by many detergent and solvents such as DMSO and thiol-containing chemicals
Assay accuracy with use of cell lysates Excellent Poor, due to cellular endopeptidase interference
Amount of Purified Enzymes Required for Inhibitor Screening 5 to 20 ng per assay point 50 to 200 ng per assay point
Amount of Substrate Required for Assay <0.5 µM per assay point 10,000 µM per assay point
Reliability of in vivo enzyme inhibition assay Excellent Poor
Accuracy of Enzyme Inhibition Assay High: direct detection of amount change of end product; not affected by chemical reagents in the assay Low: indirect detection of endopeptidase cleaved peptide; severely affected by a variety of chemical reagents in the assay 


User Guide & MSDS

[User Guide]*
*Always use the actual User Guide that shipped with your product. Is the above file locked? You can also request user guides by emailing info@epigentek.com along with your contact information and institution name.

[Safety Data Sheet]
Product Citations

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Liu J et. al. (August 2019). Inhibition of histone deacetylase protects the damaged cataract via regulating the NF-κB pathway in cultured lens epithelial cells European Journal of Inflammation. 17

Rossaert E et. al. (July 2019). Restoration of histone acetylation ameliorates disease and metabolic abnormalities in a FUS mouse model. Acta Neuropathol Commun. 7(1):107.

Nashine S et. al. (June 2019). Age-related macular degeneration (AMD) mitochondria modulate epigenetic mechanisms in retinal pigment epithelial cells. Exp Eye Res. :107701.

Zhao L et. al. (June 2019). Effects of ginsenoside Rg3 on epigenetic modification in ovarian cancer cells. Oncol Rep. 41(6):3209-3218.

Li X et. al. (May 2019). Exercise enhances the expression of brain-derived neurotrophic factor in the hippocampus accompanied by epigenetic alterations in senescence-accelerated mice prone 8. Neurosci Lett.

Chang-Shun L et. al. (April 2019). Gegen Qinlian Decoction Treats Diarrhea in Piglets by Modulating Gut Microbiota and Short-Chain Fatty Acids Front. Microbiol.

Gonneaud A et. al. (March 2019). HDAC1 and HDAC2 independently regulate common and specific intrinsic responses in murine enteroids. Sci Rep. 9(1):5363.

Liu E et. al. (March 2019). Enriched gestation activates the IGF pathway to evoke embryo-adult benefits to prevent Alzheimer's disease. Transl Neurodegener. 8:8.

Inoue K et. al. (February 2019). Podocyte histone deacetylase activity regulates murine and human glomerular diseases. J Clin Invest.

Mitsiogianni M et. al. (February 2019). Allyl isothiocyanate regulates lysine acetylation and methylation marks in an experimental model of malignant melanoma. Eur J Nutr.

Smith ER et. al. (January 2019). TGF-β1 modifies histone acetylation and acetyl-coenzyme A metabolism in renal myofibroblasts. Am J Physiol Renal Physiol.

Santamarina AB et. al. (December 2018). Supplementation of Juçara Berry (Euterpe edulis Mart.) Modulates Epigenetic Markers in Monocytes from Obese Adults: A Double-Blind Randomized Trial. Nutrients. 10(12)

Li J et. al. (November 2018). The prevention of a high dose of vitamin D or its combination with sulforaphane on intestinal inflammation and tumorigenesis in Apc<sup>1638N</sup> mice fed a high-fat diet. Mol Nutr Food Res. :e1800824.

Manoharlal et. al. (September 2018). Analysis of DNA methylome and transcriptome profiling following Gibberellin A3 (GA3) foliar application in Nicotiana tabacum L. Indian Journal of Plant Physiology. :1-14.

Wójcikowska B et al. et. al. (September 2018). Trichostatin A Triggers an Embryogenic Transition in Arabidopsis Explants via an Auxin-Related Pathway Front. Plant Sci..

Hiroshi Maejima et. al. (July 2018). Exercise and low-level GABAA receptor inhibition modulate locomotor activity and the expression of BDNF accompanied by changes in epigenetic regulation in the hippocampus Neuroscience Letters.

Rudzinskas SA et. al. (June 2018). Methamphetamine Alters DNMT and HDAC Activity in the Posterior Dorsal Medial Amygdala in an Ovarian Steroid-Dependent Manner. Neurosci Lett.

Bombardo M et. al. (June 2018). Inhibition of class I histone deacetylases abrogates TGFβ expression and development of fibrosis during chronic pancreatitis. Mol Pharmacol.

Manoharlal et. al. (May 2018). Dissecting the transcriptional networks underlying the gibberellin response in Nicotiana tabacum Biol Plant. :1-16.

Bose C et. al. (March 2018). Sulforaphane potentiates anticancer effects of doxorubicin and attenuates its cardiotoxicity in a breast cancer model. PLoS One. 13(3):e0193918.

Yang Y et. al. (February 2018). Fucoxanthin Elicits Epigenetic Modifications, Nrf2 Activation and Blocking Transformation in Mouse Skin JB6 P+ Cells. AAPS J. 20(2):32.

Halim et. al. (December 2017). Abscisic acid and salinity stress induced somaclonal variation and increased histone deacetylase (HDAC) activity in Ananas comosus var. MD2 Plant Cell Tiss Organ Cult. 2017:1-13.

Maejima H et. al. (November 2017). Exercise enhances cognitive function and neurotrophin expression in the hippocampus accompanied by changes in epigenetic programming in senescence-accelerated mice. Neurosci Lett.

Griffin Jr. E. A et al. et. al. (November 2017). Prior alcohol use enhances vulnerability to compulsive cocaine self-administration by promoting degradation of HDAC4 and HDAC5 Science Advances. 3(11)

Griffin EA Jr et. al. (November 2017). Prior alcohol use enhances vulnerability to compulsive cocaine self-administration by promoting degradation of HDAC4 and HDAC5. Sci Adv. 3(11):e1701682.

Jiang Q et. al. (March 2017). AcuC, a histone deacetylase, contributes to the pathogenicity of Aeromonas hydrophila. Microbiologyopen.

Ignácio ZM et. al. (March 2017). Quetiapine treatment reverses depressive-like behavior and reduces DNA methyltransferase activity induced by maternal deprivation. Behav Brain Res. 320:225-232.

Yang Y et. al. (March 2017). Epigenetic CpG Methylation of the Promoter and Reactivation of the Expression of GSTP1 by Astaxanthin in Human Prostate LNCaP Cells. AAPS J. 19(2):421-430.

Wawrzyniak P et. al. (January 2017). Regulation of bronchial epithelial barrier integrity by type 2 cytokines and histone deacetylases in asthmatic patients. J Allergy Clin Immunol. 139(1):93-103.

Amul Bhalodi et. al. (October 2016). Alternative Therapeutic Approach to Urothelial Cell Carcinoma with Medicinal Mushroom Extracts JCRT. 4(5):73-79.

Montagud-Romero S et. al. (May 2016). Up-regulation of histone acetylation induced by social defeat mediates the conditioned rewarding effects of cocaine. Prog Neuropsychopharmacol Biol Psychiatry.

Zhang S et. al. (October 2015). Synthesis, Biological Evaluation, and Computer-Aided Drug Designing of New Derivatives of Hyperactive Suberoylanilide Hydroxamic Acid Histone Deacetylase Inhibitors. Chem Biol Drug Des. 86(4):795-804.

Borutinskaitė V et. al. (August 2015). The Histone Deacetylase Inhibitor BML-210 Influences Gene and Protein Expression in Human Promyelocytic Leukemia NB4 Cells via Epigenetic Reprogramming. Int J Mol Sci. 16(8):18252-69.

Nio K et. al. (June 2015). Defeating EpCAM+ liver cancer stem cells by targeting chromatin remodeling enzyme CHD4 in human hepatocellular carcinoma. J Hepatol.

Nghia NA et. al. (April 2015). Long-term imipramine treatment increases N-methyl-d-aspartate receptor activity and expression via epigenetic mechanisms. Eur J Pharmacol. 752:69-77.

Passacquale G et. al. (March 2015). Aspirin-induced histone acetylation in endothelial cells enhances synthesis of the secreted isoform of netrin-1 thus inhibiting monocyte vascular infiltration. Br J Pharmacol.

Zhu Y et. al. (February 2015). Alteration of Histone Acetylation Pattern during Long-Term Serum-Free Culture Conditions of Human Fetal Placental Mesenchymal Stem Cells. PLoS One. 10(2):e0117068.

Wang L et. al. (November 2014). Blocking of JB6 Cell Transformation by Tanshinone IIA: Epigenetic Reactivation of Nrf2 Antioxidative Stress Pathway. AAPS J. 16(6):1214-25.

Saldanha SN et. al. (May 2014). Molecular mechanisms for inhibition of colon cancer cells by combined epigenetic-modulating epigallocatechin gallate and sodium butyrate. Exp Cell Res. 324(1):40-53.

Zhang W et. al. (May 2014). Histone modifications contribute to cellular replicative and hydrogen peroxide-induced premature senescence in human embryonic lung fibroblasts. Free Radic Res. 48(5):550-9.

Tiao MM et. al. (April 2014). Melatonin in the Regulation of Liver Steatosis following Prenatal Glucocorticoid Exposure. Biomed Res Int. 2014:942172.

Chaturvedi P et. al. (April 2014). Differential regulation of DNA methylation versus histone acetylation in cardiomyocytes during HHcy in vitro and in vivo: an epigenetic mechanism. Physiol Genomics. 46(7):245-55.

Siuda D et. al. (April 2014). Social isolation-induced epigenetic changes in midbrain of adult mice. J Physiol Pharmacol. 65(2):247-55.

Su ZY et. al. (March 2014). Requirement and epigenetics reprogramming of Nrf2 in suppression of tumor promoter TPA-induced mouse skin cell transformation by sulforaphane. Cancer Prev Res (Phila). 7(3):319-29.

Degen M et. al. (December 2013). Alternative therapeutic approach to renal-cell carcinoma: induction of apoptosis with combination of vitamin K3 and D-fraction. J Endourol. 27(12):1499-503.

Degen M et. al. (November 2013). Alternative Therapeutic Approach to Renal Cell Carcinoma: Induction of Apoptosis with Combination of Vitamin K3 and D-fraction. J Endourol.

Kirpich I et. al. (November 2013). Binge ethanol-induced HDAC3 down-regulates Cpt1α expression leading to hepatic steatosis and injury. Alcohol Clin Exp Res. 37(11):1920-9.

Nayebosadri A et. al. (August 2013). Endothelial nuclear lamina is not required for glucocorticoid receptor nuclear import but does affect receptor-mediated transcription activation. Am J Physiol Cell Physiol. 305(3):C309-22.

Jeong JB et. al. (June 2013). Patchouli alcohol, an essential oil of Pogostemon cablin, exhibits anti-tumorigenic activity in human colorectal cancer cells. Int Immunopharmacol. 16(2):184-90.

Yuanyuan Deng et. al. (January 2013). Z-GP Modification to CI-994, a Histone Deacetylase Inhibitor and the Application in Targeting Carcinoma Chemotherapy Medicinal chemistry. 3(2):199-205. Full Article

Yang J et. al. (January 2012). Stem cell gene SALL4 suppresses transcription through recruitment of DNA methyltransferases. J Biol Chem. 287(3):1996-2005.

Customer Reviews

Rating by e**************@yahoo.com Unverified Purchase Reviewed on: Thursday 31 August, 2017
Application Description
HDAC activity in nuclear lysates - used three assays from three companies

Procedural Details
As per instructions- split the HDAC nuclear lysates into three - as per manufacture instructions...

Other Thoughts
Somethings off--- the fluorometric assays , have a very strong inhibition - as reflected in the output values. This kit - same, enzyme, same inhibitors- same concentration - much weaker inhibition. Other compounds ,not HDAC inhibitors - are very strong with this particular assay - but when confirmed with regular assays- the do NOT inhibit the enzyme. Either this kit has flaws OR, the other kits have flaws.. independent and more than false positive issues.
Rating by R***************@ Unverified Purchase Reviewed on: Monday 18 July, 2016
Application Description
We do work on gene and protein expression changes (epigenetic modulators) in neuroglia differentiation and behaviour. This product was used to measure HDAC activity/inhibition in samples treated with HDAC inhibitor.

Procedural Details
HDAC activity/inhibition

Starting Material

Protocol Overview
Protocol followed as per the user guide provided by the company

Store the reagents as suggested in user guide, reagents prepared freshly while using take more protein(5-10ug), after adding the SS solution take reading as soon as possible

Results Summary
In HDAC inhibitor treated samples the HDAC activity is less compared to the untreated control.

Other Thoughts
Make the purified enzyme or nuclear extract ready before using the kit, make the required dilutions of HDAC inhibitors.

The Good
Easy to handle

The Bad
Cost, HDAC enzyme or nuclear extract is not supplied to screen the HDACis

Review Source: http://www.biocompare.com/Product-Reviews/173539-To-measure-the-HDAC-activity-Inhibition/
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