Science Catalog 2012 Life Cell Signaling Histone Deacetylase Assays HDAC-Glo™ I/II Assays and Screening Systems Product HDAC-Glo™ I/II Assay HDAC-Glo™ I/II Screening System Product HDAC-Glo™ I/II Control Substrate HeLa Nuclear Extract Trichostatin A Size Cat.# Price (Fr) 10 ml G6420 578.00 Size Conc. 10 µl For Research Use Only. Not for Use in Diagnostic Procedures. Description: The HDAC-Glo™ I/II Assays and Screening Systems are single-reagentaddition, homogeneous, luminescent assays that measure the relative activity of histone deacetylase (HDAC) class I and II enzymes from cells, extracts or purified enzyme sources. The assays use an acetylated, live-cell-permeant, luminogenic peptide substrate that can be deacetylated by HDAC activities. Deacetylation of the peptide aminoluciferin substrate is measured using a coupled enzymatic system in which a protease in the Developer Reagent cleaves the deacetylated peptide from the aminoluciferin substrate, releasing aminoluciferin, which is quantified in a reaction using Ultra-Glo™ recombinant firefly luciferase. The assay reaction is typically complete within 15–45 minutes with no sample manipulation. The HDAC-mediated luminescent signal is persistent, with a half-life of greater than 3 hours, allowing batch processing of multiwell plates. The HDAC assay is broadly useful for class I and II enzymes. The Trichostatin A, included in the HDAC-Glo™ I/II Screening Systems or available separately, is a known pan HDAC inhibitor that may be used as a positive control inhibitor. The Trichostatin A is supplied at a concentration of 10mM in DMSO. The HeLa Nuclear Extract, included in the HDAC-Glo™ I/II Screening Systems or available separately, may be used as a source of histone deacetylase activity. The diluted extract also can be used as an HDAC-Glo™ I/II Assay chemistry control. The HDAC-Glo™ I/II Control Substrate, only available separately, is a nonacetylated form of the HDAC-Glo™ I/II Substrate with the same amino acid sequence and can be used with the HDAC-Glo™ I/II Assays and Screening Systems to confirm true HDAC inhibition in secondary screens. The Control Substrate is supplied at a concentration of 10mM and is sufficient for 480 assays in 96-well plate format when combined with the HDAC-Glo™ Reagent prepared with components in the HDAC-Glo™ I/II Assays or Screening Systems. 5 × 10 ml G6421 2168.00 100 ml G6422 3468.00 10 ml G6430 615.00 5 × 10 ml G6431 2307.00 Cat.# Price (Fr) G6550 353.00 10 µl 5 mg/ml G6570 165.00 10 µl 10 mM G6560 143.00 Features: • Simple Measurement of Deacetylating Activities: Use a singlereagent-addition, homogeneous, add-mix-measure protocol for easy implementation from benchtop to screening. • Highly Sensitive: Obtain 10- to 100-fold higher sensitivity than comparable fluorescence methods. • Fast Data Acquisition: Achieve maximum signal in as little as 15 minutes with persistent glow-type steady-state signal, making the protocol amenable to automation in high-throughput formats and compatible with luminometers without injectors. • Flexible to Sample Type: Use with viable cells, extracts or purified recombinant enzyme sources. Storage Conditions: Store the HDAC-Glo™ Assay components and HDAC-Glo™ I/II Control Substrate (sold separately) at –20°C. Store HeLa Nuclear Extract at –70°C. Protocol HDAC-Glo™ I/II Assay and Screening System Technical Manual 100,000 10,000 1,000 100 10 1 1 Part# TM335 HDAC 1 HDAC 2 HDAC 3 HDAC 8 HDAC 4 HDAC 5 HDAC 7 HDAC 9 HDAC 6 HDAC 10 HDAC 11 10 100 HDAC Enzyme (ng/ml) Broad linearity with HDAC Class I and II enzymes. 1,000 52 For complete and up-to-date product information visit: www.promega.com/catalog Signal-to-Noise Ratio 8907MA Pagina 55
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