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.
Product Specification
| Quantity: | 96 assays |
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| 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, dilutei n 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°C1/2 volume microplate (Prod. No. BML-KI101) Storage: Room temperature 1/2 volume white microplate (Prod. No. BML-KI110) Storage: Room temperature |
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| Shipping: | SHIPPED ON DRY ICE |
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| Long Term Storage: | -80°C |
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| 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. BMLKI-104, 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. |
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| Miscellaneous/General: | HDACs are typically found in multiprotein complexes and are tightly regulated by subcellular localization, phosphorylation, and likely by other mechanisms. |
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| Background / Technical Information: | Please click here for the comprehensive product datasheet. |
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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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General Literature References
Acetylation: a regulatory modification to rival phosphorylation? T. Kouzarides et al. Embo J. 19 1176 (2000)
AbstractHistone deacetylase inhibitors arrest polyglutamine-dependent neurodegeneration in Drosophila J.S. Steffan et al. Nature 413 739 (2001)
AbstractSmall molecule activators of sirtuins extend Saccharomyces cerevisiae lifespan K.T. Howitz Nature 425 191 (2003)
AbstractSuberoylanilide 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)
AbstractA novel action of histone deacetylase inhibitors in a protein aggresome disease model L.J. Corcoran et al. Curr. Biol. 14 488 (2004)
AbstractHistone deacetylase inhibitors P.A. Marks et al. Adv. Cancer Res. 91 137 (2004)
AbstractIncreased nuclear NAD biosynthesis and SIRT1 activation prevent axonal degeneration T. Araki et al. Science 305 1010 (2004)
AbstractModulation of NF-kappaB-dependent transcription and cell survival by the SIRT1 deacetylase F. Yeung et al. Embo J. 23 2369 (2004)
AbstractSirt1 promotes fat mobilization in white adipocytes by repressing PPAR-gamma F. Picard et al. Nature 429 771 (2004)
AbstractSirtuin activators mimic caloric restriction and delay ageing in metazoans J.G. Wood et al. Nature 430 686 (2004)
AbstractTargeting CREB-bindinag protein (CBP) loss of function as a therapeutic strategy in neurological disorders C. Rouaux et al. Biochem. Pharmacol. 68 1157 (2004)
AbstractThe novel histone deacetylase inhibitor BL1521 inhibits proliferation and induces apoptosis in neuroblastoma cells A.J. de Ruijter Biochem Pharmacol 68 1279 (2004)
AbstractPhase 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)
AbstractPhase 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)
AbstractResveratrol rescues mutant polyglutamine cytotoxicity in nematode and mammalian neurons J.A. Parker et al. Nat. Genet. 37 349 (2005)
AbstractSIRT1 protects against microglia-dependent amyloid-beta toxicity through inhibiting NF-kappaB signaling J. Chen et al. J. Biol. Chem. 280 40364 (2005)
AbstractNeuronal protection by sirtuins in Alzheimer's disease T.S. Anekonda et al. J Neurochem 96 305 (2006)
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