Useful for assaying lysates, immunoprecipitates or inhibitor screening using the nuclear extract provided
Includes HeLa nuclear extract, a rich source of HDACs 1 & 2 for use as a positive control or as a source of HDAC activity for screening
Compatible with class I & IIb HDAC and sirtuins (with addition of NAD+)
Includes enough reagent for 100-200 assays
No radioactivity. No extractions. HTS friendly-mix and read on one 96-well plate. For class I and class II HDACs/sirtuins. Applications include cell-based assays and assay of immunoprecipitates.
Histone deacetylase inhibitors have shown promise as anti-tumor agents and naturally this has stimulated interest in the screening of compounds for HDAC inhibition. The FLUOR DE LYS® HDAC fluorometric activity assay kit is a sensitive and convenient alternative to protocols utilizing radiolabeled, acetylated histones or peptide/HPLC methods for the assay of histone deacetylases. It is based on the unique FLUOR DE LYS® (Fluorimetric Histone deAcetylaseLysyl) substrate and developer combination and provides an assay that can be carried out in two simple mixing steps, all on the same 96-well plate. First, the FLUOR DE LYS® substrate which comprises an acetylated lysine side chain, is incubated with a sample containing HDAC activity (HeLa nuclear or other extract, purified enzyme, bead bound immunocomplex, etc.). Deacetylation of the substrate sensitizes the substrate so that, in the second step, mixing with the FLUOR DE LYS® developer generates a fluorophore. The assay has been used successfully with preparations of all the known class I HDACs-HDAC1, HDAC2, HDAC3 and HDAC8 (see product data sheet) with class II HDACs 4-7, 9 and 10 and with the human Sir2 homolog, SIRT1 (see product data sheet). Work at Enzo Life Sciences has shown that the FLUOR DE LYS® substrate is cell-permeable and is deacetylated in situ by cellular HDACs. The deacetylated substrate accumulates inside cells and may be quantified by addition of FLUOR DE LYS® developer to a cell lysate.
Figure 1: Reaction Scheme of the HDAC Fluorescent Activity Assay. Deacetylation of the substrate sensitizes it to the developer, which then generates a fluorophore (symbol). The fluorophore is excited with 360 nm light and the emitted light (460 nm) is detected on a fluorometric plate reader.
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Product Details
Alternative Name:
Histone deacetylase fluorescent assay kit
Applications:
Fluorescent detection, HTS Activity assay
Use/Stability:
Store all components, except the microtiter plate, at -80°C for the highest stability. The HeLa Nuclear Extract, BML-KI140, must be handled with particular care in order to retain maximum enzymatic activity. Defrost it quickly in a RT water bath or by rubbing between fingers, then immediately store on an ice bath. The remaining unused extract should be refrozen quickly, by placing at -80°C. If possible, snap freeze in liquid nitrogen or a dry ice/ethanol bath. To minimize the number of freeze/thaw cycles, aliquot the extract into separate tubes and store at -80°C. The FLUOR DE LYS® Substrate, BML-KI104, when diluted in Assay Buffer, may precipitate after freezing and thawing. It is best, therefore, to dilute only the amount needed to perform the assays of that day.
Shipping:
Dry Ice
Long Term Storage:
-80°C
Contents:
Nuclear Extract from HeLa Cells (human cervical cancer cell line) (Prod. No. BML-KI140-0100)
(100 µl; In 0.1M KCl, 20mM HEPES/NaOH, pH 7.9, 20% (v/v) glycerol, 0.2mM EDTA, 0.5mM DTT, 0.5Mm PMSF, prepared according to a modification of J.D. Dignam et al. (1983) and S.M. Abmayr et al. (1988)).
Storage: -80°C, avoid freeze/thaw cycles FLUOR DE LYS® Substrate (Prod. No. BML-KI104-0050)
(50 µl; 50mM in DMSO)
Storage: -80°C FLUOR DE LYS® Developer Concentrate (20x) (Prod. No. BML-KI105-0300) (300 µl; 20x stock solution, dilute in assay buffer before use)
Storage: -80°C Trichostatin A (HDAC Inhibitor) (Prod. No. BML-GR309-9090)
(100 µl; 0.2mM in DMSO)
Storage: -80°C FLUOR DE LYS® Deacetylated Standard (Prod. No. BML-KI142-0030)
(30 µl; 10mM in DMSO)
Storage: -80°C HDAC Assay Buffer (Prod. No. BML-KI143-0020)
(20 ml; 50mM TRIS/Cl, pH 8.0, 137mM NaCl, 2.7mM KCl, 1mM MgCl2)
Storage: -20°C 1/2 volume microplate (Prod. No. BML-KI101)
Storage: Ambient 1/2 volume white microplate (Prod. No. BML-K571)
Storage: Ambient
Regulatory Status:
RUO - Research Use Only
Product Literature References
The activation of histone deacetylases 4 prevented endothelial dysfunction: A crucial mechanism of HuangqiGuizhiWuwu Decoction in improving microcirculation dysfunction in diabetes: M. Chen, et al.; J. Ethnopharmacol. 307, 116240 (2023), Abstract;
Four New Anthraquinones with Histone Deacetylase Inhibitory Activity from Ventilago denticulata Roots: N. Hangsamai, et al.; Molecules 27, 1088 (2022), Abstract;
Ulleunganilines A–C, Trichostatin Analogues Bearing a Modified Side Chain from Streptomyces sp. 13F051: G.J. Hwang, et al.; J. Nat. Prod. 84, 2420 (2021), Abstract;
Prolactin and Estradiol are Epigenetic Modulators in Bovine Mammary Epithelial Cells during Staphylococcus aureus Infection: M.G. Salgado-Lora, et al.; Pathogens 9, 520 (2020), Abstract; Full Text
The Contribution of Romidepsin to the Herbicidal Activity of Burkholderia rinojensis Biopesticide: D.K. Owens, et al.; J. Nat. Prod. 83, 843 (2020), Abstract;
Efficacy of Azatyrosine-Phenylbutyric Hydroxamides, a Histone Deacetylase Inhibitor, on Chemotherapy-Induced Gastrointestinal Mucositis: P.L. Liao, et al.; Int. J. Mol. Sci. 20, E249 (2019), Abstract;
Differences in Functional Expression of Connexin43 and NaV1.5 by Pan- and Class-Selective Histone Deacetylase Inhibition in Heart: X. Zhang, et al.; Int. J. Mol. Sci. 19, E2288 (2018), Abstract; Full Text
Loss of DBC1 (CCAR2) affects TNFα-induced lipolysis and Glut4 gene expression in murine adipocytes: A.A. Able, et al.; J. Mol. Endocrinol. 61, 195 (2018), Abstract;
Mechanisms involved in epigenetic down-regulation of Gfap under maternal hypothyroidism: P. Kumar, et al.; Biochem. Biophys. Res. Commun. 502, 375 (2018), Abstract;
Metformin overcomes high glucose-induced insulin resistance of podocytes by pleiotropic effects on SIRT1 and AMPK: D. Rogacka, et al.; Biochim. Biophys. Acta Mol. Basis Dis. 1864, 115 (2018), Abstract;
Monomeric and oligomeric flavanols maintain the endogenous glucocorticoid response in human macrophages in pro-oxidant conditions in vitro: G. Verissimo, et al.; Chem. Biol. Interact. 291, 237 (2018), Abstract;
SIRT1/FoxO3 axis alteration leads to aberrant immune responses in bronchial epithelial cells: S. Di Vincenzo, et al.; J. Cell. Mol. Med. 22, 2272 (2018), Abstract; Full Text
Sulforaphane restores acetyl-histone H3 binding to Bcl-2 promoter and prevents apoptosis in ethanol-exposed neural crest cells and mouse embryos: F. Yuan, et al.; Exp. Neurol. 300, 60 (2018), Abstract;
The first-in-class alkylating deacetylase inhibitor molecule tinostamustine shows antitumor effects and is synergistic with radiotherapy in preclinical models of glioblastoma: C. Festuccia, et al.; J. Hematol. Oncol. 11, 32 (2018), Abstract; Full Text
Antiproliferative effects of TSA, PXD‑101 and MS‑275 in A2780 and MCF7 cells: Acetylated histone H4 and acetylated tubulin as markers for HDACi potency and selectivity: V.P. Androutsopoulos, et al.; Oncol. Rep. 38, 3412 (2017), Abstract; Full Text
Design, synthesis and evaluation of novel N-hydroxybenzamides/N-hydroxypropenamides incorporating quinazolin-4(3H)-ones as histone deacetylase inhibitors and antitumor agents: D.T. Hieu, et al.; Bioorg. Chem. 76, 258 (2017), Abstract;
SIRT4 Is a Lysine Deacylase that Controls Leucine Metabolism and Insulin Secretion: K.A. Anderson, et al.; Cell Metab. 25, 838 (2017), Abstract; Full Text
Adiponectin corrects premature cellular senescence and normalizes antimicrobial peptide levels in senescent keratinocytes: T. Jin, et al.; Biochem. Biophys. Res. Commun. 16, 31037 (2016), Application(s): Deacetylation activity measurement, Abstract;
Hybrid Enzalutamide Derivatives with Histone Deacetylase Inhibitor Activity Decrease Heat Shock Protein 90 and Androgen Receptor Levels and Inhibit Viability in Enzalutamide-Resistant C4-2 Prostate Cancer Cells: R. Rosati, et al.; Mol. Pharmacol. 90, 225 (2016), Abstract; Full Text
Identification of new quinic acid derivatives as histone deacetylase inhibitors by fluorescence-based cellular assay: D. Son, et al.; Bioorg. Med. Chem. Lett. 26, 2365 (2016), Abstract;
Investigating the Sensitivity of NAD+-Dependent Sirtuin Deacylation Activities to NADH: A.S. Madsen, et al.; J. Biol. Chem. 291, 7128 (2016), Abstract; Full Text
Reversible Glutathionylation of Sir2 by Monothiol Glutaredoxins Grx3/4 Regulates Stress Resistance: N. Vall-Llaura, et al.; Free Radic. Biol. Med. 96, 45 (2016), Application(s): In vitro Sir2 activity was assayed, Abstract;
Synthesis, biological characterization and molecular modeling insights of spirochromanes as potent HDAC inhibitors: F. Thaler, et al.; Eur. J. Med. Chem. 108, 53 (2016), Abstract;
Wnt Protein Signaling Reduces Nuclear Acetyl-CoA Levels to Suppress Gene Expression during Osteoblast Differentiation: C.M. Karner, et al.; J. Biol. Chem. 291, 13028 (2016), Abstract; Full Text
Hybrids from 4-anilinoquinazoline and hydroxamic acid as dual inhibitors of vascular endothelial growth factor receptor-2 and histone deacetylase: F.W. Peng, et al.; Bioorg. Med. Chem. Lett. 25, 5137 (2015), Application(s): Measurement of HDAC inhibitory activity , Abstract;
NBM-T-BBX-OS01, Semisynthesized from Osthole, Induced G1 Growth Arrest through HDAC6 Inhibition in Lung Cancer Cells: J.T. Pai, et al.; Molecules 20, 8000 (2015), Application(s): Assay using human H1299 cell lysates, Abstract; Full Text
Screening and profiling assays for HDACs and sirtuins: K.T. Howitz; Drug Discov. Today Technol. 18, 38 (2015), Abstract;
The effect of sulforaphane on histone deacetylase activity in keratinocytes: Differences between in vitro and in vivo analyses: S.E. Dickinson, et al.; Mol. Carcinog. 54, 1513 (2015), Abstract; Full Text
Molecular mechanism of sphingosine-1-phosphate action in Duchenne muscular dystrophy: D.H. Nguyen-Tran, et al.; Dis. Model Mech. 7, 41 (2014), Application(s): Assay, Abstract; Full Text
NL-103, a novel dual-targeted inhibitor of histone deacetylases and hedgehog pathway, effectively overcomes vismodegib resistance conferred by Smo mutations: J. Zhao, et al.; Pharmacol. Res- Perspect. 2, e00043 (2014), Abstract; Full Text
Synthesis and anticancer activities of thieno[3,2-d]pyrimidines as novel HDAC inhibitors: Q. Tan, et al.; Bioorg. Med. Chem. 22, 358 (2014), Application(s): Assay, Abstract;
Benzofused hydroxamic acids: useful fragments for the preparation of histone deacetylase inhibitors. Part 1: hit identification: E. Marastoni, et al.; Bioorg. Med. Chem. Lett. 23, 4091 (2013), Application(s): Assay, Abstract;
Design, synthesis and biological evaluation of di-substituted cinnamic hydroxamic acids bearing urea/thiourea unit as potent histone deacetylase inhibitors: C. Ning, et al.; Bioorg. Med. Chem. Lett. 23, 6432 (2013), Application(s): Assay, Abstract;
Dual-Acting Histone Deacetylase-Topoisomerase I Inhibitors: W. Guerrant, et al.; Bioorg. Med. Chem. Lett. 23, 3283 (2013), Application(s): Assay, Abstract; Full Text
Efficient new constructs against triple negative breast cancer cells: synthesis and preliminary biological study of ferrocifen-SAHA hybrids and related species: J.D.J. Cazares Marinero, et al.; Dalton Trans. 42, 15489 (2013), Abstract;
Novel N-hydroxyfurylacrylamide-based histone deacetylase (HDAC) inhibitors with branched CAP group (Part 2): T. Feng, et al.; Bioorg. Med. Chem. 21, 5339 (2013), Abstract;
Rejuvenating sirtuins: the rise of a new family of cancer drug targets: S. Bruzzone, et al.; Curr. Pharm. Des. 19, 614 (2013), Abstract; Full Text
A novel series of l-2-benzyloxycarbonylamino-8-(2-pyridyl)-disulfidyloctanoic acid derivatives as histone deacetylase inhibitors: design, synthesis and molecular modeling study: D. Huang, et al.; Eur. J. Med. Chem. 52, 111 (2012), Abstract;
Lactate, a product of glycolytic metabolism, inhibits histone deacetylase activity and promotes changes in gene expression: T. Latham, et al.; Nucleic Acids Res. 40, 4794 (2012), Abstract; Full Text
Synthesis, evaluation and molecular modeling of cyclic tetrapeptide histone deacetylase inhibitors as anticancer agents: D. Huang, et al.; J. Pep. Sci. 18, 242 (2012), Abstract;
Activated microglia decrease histone acetylation and Nrf2-inducible anti-oxidant defence in astrocytes: restoring effects of inhibitors of HDACs, p38 MAPK and GSK3β: F. Correa, et al.; Neurobiol. Dis. 44, 142 (2011), Application(s): HDAC activity in astrocytes, Abstract;
Heparanase-mediated loss of nuclear syndecan-1 enhances histone acetyltransferase (HAT) activity to promote expression of genes that drive an aggressive tumor phenotype: A. Purushothanman, et al.; J. Biol. Chem. 286, 30377 (2011), Abstract; Full Text
Identification of dehydroxytrichostatin A as a novel up-regulator of the ATP-binding cassette transporter A1 (ABCA1): Y. Xu, et al.; Molecules 16, 7183 (2011), Abstract; Full Text
Nuclear import of histone deacetylase 5 by requisite nuclear localization signal phosphorylation: T.M. Greco, et al.; Mol. Cell. Proteomics 10, M110.004317 (2011), Application(s): Activity of immunoprecipitated HDAC5, Abstract; Full Text
Rescue of the mutant CFTR chloride channel by pharmacological correctors and low temperature analyzed by gene expression profiling: E. Sondo, et al.; Am. J. Physiol. Cell. Physiol. 301, C872 (2011), Application(s): HDAC activity in lung cell lines, Abstract; Full Text
Drosophila SIN3 isoforms interact with distinct proteins and have unique biological functions: M.M. Spain, et al.; J. Biol. Chem. 285, 27457 (2010), Application(s): HDAC activity in nuclear extracts from S2 Drosophila cells, Abstract; Full Text
HDAC3 is negatively regulated by the nuclear protein DBC1: C.C. Chini, et al.; J. Biol. Chem. 285, 40830 (2010), Abstract; Full Text
Sulforaphane Retards the Growth of Human PC-3 Xenografts and Inhibits HDAC Activity in Human Subjects: M.C. Myzak, et al.; Exp Biol Med 232, 227 (2007), Application(s): HDAC activity in PBMC lysates, Abstract; Full Text
Sulforaphane inhibits histone deacetylase in vivo and suppresses tumorigenesis in Apc-minus mice: M.C. Myzak, et al.; FASEB J. 20, 506 (2006), Application(s): HDAC activity in PBMC lysates, Abstract; Full Text
Histone deacetylase is a target of valproic acid-mediated cellular differentiation: N. Gurvich et al.; Cancer Res. 64, 1079 (2004), Abstract;
Phosphorus-based SAHA analogues as histone deacetylase inhibitors: G.V. Kapustin et al.; Org. Lett. 5, 3053 (2003), Abstract;
Inhibition of silencing and accelerated aging by nicotinamide, a putative negative regulator of yeast sir2 and human SIRT1: K.J. Bitterman et al.; J. Biol. Chem. 277, 45099 (2002), Abstract;
Cloning and characterization of a histone deacetylase, HDAC9: X. Zhou et al.; PNAS 98, 10572 (2001), Abstract;
General Literature References
Coupling of histone deacetylation to NAD breakdown by the yeast silencing protein Sir2: Evidence for acetyl transfer from substrate to an NAD breakdown product: J.C. Tanny et al.; PNAS 98, 415 (2001), Abstract;
A phylogenetically conserved NAD+-dependent protein deacetylase activity in the Sir2 protein family: J.S. Smith et al.; PNAS 97, 6658 (2000), Abstract;
Acetylation and chromosomal functions: W.L. Cheung et al.; Curr. Opin. Cell Biol. 12, 326 (2000), Abstract;
Cloning and characterization of a novel human class I histone deacetylase that functions as a transcription repressor: E. Hu et al.; J. Biol. Chem. 275, 15254 (2000), Abstract;
Histone deacetylases: silencers for hire: H.H. Ng et al.; Trends Biochem. Sci. 25, 121 (2000), Abstract;
Isolation of a novel histone deacetylase reveals that class I and class II deacetylases promote SMRT-mediated repression: H.-Y. Kao et al.; Genes Dev. 14, 55 (2000), Abstract;
Role of NAD(+) in the deacetylase activity of the SIR2-like proteins: J. Landry et al.; BBRC 278, 685 (2000), Abstract;
Silent information regulator 2 family of NAD- dependent histone/protein deacetylases generates a unique product, 1-O-acetyl-ADP-ribose: K.G. Tanner et al.; PNAS 97, 14178 (2000), Abstract;
The language of covalent histone modifications: B.D. Strahl et al.; Nature 403, 41 (2000), Abstract;
Transcriptional silencing and longevity protein Sir2 is an NAD-dependent histone deacetylase: S. Imai et al.; Nature 403, 795 (2000), Abstract;
