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FLUOR DE LYS® HDAC fluorometric cellular activity assay kit

Monitor HDAC activity in cell culture using cell-permeable FLUOR DE LYS® substrate
BML-AK503-0001 96 wells 423.00 USD
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The HDAC fluorometric cellular activity assay allows the determination of deacetylase activity within an undisturbed cellular environment and provides accurate activity information that is reflective of endogenous regulation. It also allows the study of the effects of upstream regulators on deacetylase activity and the detection of inhibitors or activators that act indirectly to affect deacetylase activity. The FLUOR DE LYS® HDAC substrate is cell permeable. It is deacetylated by cellular HDACs and thus suitable for high-throughput cell-based deacetylase assays. This kit provides the reagents and protocols needed for determining rates of intracellular deacetylase activity with cultured cells. Deacetylation can be quantitated by addition of developer to the media and lysed cells.
BML-AK503 fluordelys
Figure: Method for Assaying Intracellular HDAC Activity with the Fluorogenic, Cell-permeable Substrate, FLUOR DE LYS® (Prod. No. BML-KI104). FLUOR DE LYS® is added to cell growth medium and enters the cells. Deacetylation by class I/II HDACs or sirtuins yields the deacetylated form of FLUOR DE LYS®, some of which may exit the cell. Lysis of the cells with detergent allows contact between the non-cell permeable Developer (Prod. No. BML-KI105) and both intra- and extracellular deacetylated substrate, thus producing a fluorescent signal (symbol). A strong class I/II HDAC inhibitor (e.g. trichostatin A) is added along with the lysis buffer containing detergent to insure that no deacetylation occurs after cell lysis.
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BML-AK503 fluordelys

Product Specification

Alternative Name:Cellular Histone deacetylase fluorescent assay kit
Applications:Fluorescent detection, HTS
Activity assay, Cell-based assays
Use/Stability:Store all components except the microplate and instruction booklet at -70°C for the highest stability. The HeLa Nuclear Extract, Prod. No. BML-KI345, 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 -70°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 -70°C. The FLUOR DE LYS® Substrate, Prod. No. 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.
Long Term Storage:-80°C
Kit/Set Contains:Nuclear Extracts from Hela Cells (human cervical cancer cell line) (Prod. No. BML-KI345)
(50 µl; 2 mg protein/ml in 0.1M potassium chloride, 20mM HEPES/sodium hydroxide, pH 7.9, 20% (v/v) glycerol, 0.2mM ethylenediaminetetraacetic acid, 0.5M dithiothreitol, 0.1mM PMSF; Prepared according to a midification of J.D. Dignam et al (1983) and S.M. Abmayr et al. (1988))
Storage: -70°C, avoid freeze/thaw cycles!
FLUOR DE LYS® Substrate (Prod. No. BML-KI104)
(50 µl; 50mM in DMSO)
Storage: -70°C
FLUOR DE LYS® Developer Concentrate (20x)
(300 µl; 20x stock solution, dilute in assay buffer before use)
Storage: -70°C
Trichostatin A (HDAC Inhibitor) (Prod. No. BML-GR-309-9090) (100 µl; 0.2mM in DMSO) Storage: -70°C
Nicotinamide (Sirtuin Inhibitor) (Prod. No. BML-KI283) (500 µl; 50mM nicotinamide in 50mM TRIS/Cl, pH 8.0, 137mM sodium chloride, 2.7mM potassium chloride, 1mM magnesium chloride)
Storage: -70°C
FLUOR DE LYS® Deacetylated Standard (Prod. No. BML-KI142)
(30 µl; 10mM in DMSO)
Storage: -70°C
HDAC Assay Buffer (50mM TRIS/Cl, pH 8.0, 137mM sodium chloride, 2.7 mM potassium chloride, 1mM magnesium chloride) (Prod. No. BML-KI143)
(20 ml)
Storage: -70°C
Cell Lysis Buffer (Prod. No. BML-KI143)
(20 ml; 1.0% NP-40 in HDAC Assay Buffer)
Storage: -70°C
(2) 1/2 volume microplates (Prod. No. BML-KI101)
Storage: Room temperature (One sterile, One non-sterile)
Scientific Background:HDACs are typically found in multiprotein complexes and are tightly regulated by subcellular localization, phosphorylation, and likely by other mechanisms.

Product Literature References

Targeting the invasive phenotype of cisplatin-resistant Non-Small Cell Lung Cancer cells by a novel histone deacetylase inhibitor: V. Zuco, et al.; Biochem. Pharmacol. (2015), Application(s): Assay, Abstract;
The tobacco smoke component acrolein induces glucocorticoid resistant gene expression via inhibition of histone deacetylase: M.J. Randall, et al.; Toxicol. Lett. (2015), Application(s): Nuclear deacetylase activity, Abstract;
A novel valproic acid prodrug as an anticancer agent that enhances doxorubicin anticancer activity and protects normal cells against its toxicity in vitro and in vivo: N. Tarasenko, et al.; Biochem. Pharmacol. 88, 158 (2014), Abstract;
Liquid fructose downregulates Sirt1 expression and activity and impairs the oxidation of fatty acids in rat and human liver cells: A. Rebollo, et al.; Biochim. Biophys. Acta 1841, 514 (2014), Abstract;
Histone deacetylase inhibition reduces cardiac connexin43 expression and gap junction communication: Q. Xu, et al.; Front. Pharmacol. 4, 44 (2013), Application(s): HDAC activity in ventricular myocytes and HeLa cells, Abstract; Full Text
NAMPT/PBEF1 enzymatic activity is indispensable for myeloma cell growth and osteoclast activity: S.U. Venkateshaiah, et al.; Exp. Hematol. 41, 547 (2013), Application(s): SIRT1 activity in myeloma cells, Abstract;
Disparate impact of butyroyloxymethyl diethylphosphate (AN-7), a histone deacetylase inhibitor, and doxorubicin in mice bearing a mammary tumor: N. Tarasenko, et al.; PLoS One 7, e31393 (2012), Application(s): HDAC activity in a variety of cell lines, Abstract; Full Text
Hypoxia initiates sirtuin1-mediated vascular endothelial growth factor activation in choroidal endothelial cells through hypoxia inducible factor-2α: S. Balaiya, et al.; Mol. Vis. 18, 114 (2012), Abstract;
Hypoxia initiates sirtuin1-mediated vascular endothelial growth factor activation in choroidal endothelial cells through hypoxia inducible factor–2α: S. Balaiya, et al.; Mol. Vis. 18, 114 (2012), Application(s): HDAC activity in choroidal endothelial cells, Abstract; Full Text
Heparanase-mediated loss of nuclear syndecan-1 enhances histone acetyltransferase (HAT) activity to promote expression of genes that drive an aggressive tumor phenotype: A. Purushothaman, et al.; J. Biol. Chem. 286, 30377 (2011), Abstract;
Leptin boosts cellular metabolism by activating AMPK and the sirtuins to reduce tau phosphorylation and β-amyloid in neurons: S.J. Greco, et al.; BBRC 414, 170 (2011), Application(s): SIRT activity in a human neuroblastoma cell line, Abstract; Full Text
Reactivity of mouse alveolar macrophages to cigarette smoke is strain dependent: D. Vecchio, et al.; Am. J. Physiol. Lung Cell. Mol. Physiol. 298, L704 (2010), Application(s): HDAC activity in mouse alveolar macrophages, Abstract; Full Text
Romidepsin (FK228), a potent histone deacetylase inhibitor, induces apoptosis through the generation of hydrogen peroxide: H. Mizutani, et al.; Cancer Sci. 101, 2214 (2010), Application(s): HDAC activity in leukaemia cells, Abstract; Full Text

General Literature References

Neuronal protection by sirtuins in Alzheimer's disease: T.S. Anekonda et al.; J Neurochem 96, 305 (2006), Abstract;
Phase I and pharmacokinetic study of MS-275, a histone deacetylase inhibitor, in patients with advanced and refractory solid tumors or lymphoma: Q. C. Ryan et al.; J. Clin. Oncol. 23, 3912 (2005), Abstract;
Phase I study of an oral histone deacetylase inhibitor, suberoylanilide hydroxamic acid, in patients with advanced cancer: W. K. Kelly et al.; J. Clin. Oncol. 23, 3923 (2005), Abstract;
Resveratrol rescues mutant polyglutamine cytotoxicity in nematode and mammalian neurons: J.A. Parker et al.; Nat. Genet. 37, 349 (2005), Abstract;
SIRT1 protects against microglia-dependent amyloid-beta toxicity through inhibiting NF-kappaB signaling: J. Chen et al.; J. Biol. Chem. 280, 40364 (2005), Abstract;
A novel action of histone deacetylase inhibitors in a protein aggresome disease model: L.J. Corcoran et al.; Curr. Biol. 14, 488 (2004), Abstract;
Histone deacetylase inhibitors: P.A. Marks et al.; Adv. Cancer Res. 91, 137 (2004), Abstract;
Increased nuclear NAD biosynthesis and SIRT1 activation prevent axonal degeneration: T. Araki et al.; Science 305, 1010 (2004), Abstract;
Modulation of NF-kappaB-dependent transcription and cell survival by the SIRT1 deacetylase: F. Yeung et al.; Embo J. 23, 2369 (2004), Abstract;
Sirt1 promotes fat mobilization in white adipocytes by repressing PPAR-gamma: F. Picard et al.; Nature 429, 771 (2004), Abstract;
Sirtuin activators mimic caloric restriction and delay ageing in metazoans: J.G. Wood et al.; Nature 430, 686 (2004), Abstract;
Targeting CREB-bindinag protein (CBP) loss of function as a therapeutic strategy in neurological disorders: C. Rouaux et al.; Biochem. Pharmacol. 68, 1157 (2004), Abstract;
The novel histone deacetylase inhibitor BL1521 inhibits proliferation and induces apoptosis in neuroblastoma cells: A.J. de Ruijter; Biochem Pharmacol 68, 1279 (2004), Abstract;
Small molecule activators of sirtuins extend Saccharomyces cerevisiae lifespan: K.T. Howitz; Nature 425, 191 (2003), Abstract;
Suberoylanilide hydroxamic acid, a histone deacetylase inhibitor, ameliorates motor deficits in a mouse model of Huntington’s disease: E. Hockly et al.; PNAS 100, 2041 (2003), Abstract;
Histone deacetylase inhibitors arrest polyglutamine-dependent neurodegeneration in Drosophila: J.S. Steffan et al.; Nature 413, 739 (2001), Abstract;
Acetylation: a regulatory modification to rival phosphorylation?: T. Kouzarides et al.; Embo J. 19, 1176 (2000), Abstract;

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