Cell Signaling SIRT-Glo™ Assays and Screening Systems Product SIRT-Glo™ Assay SIRT-Glo™ Screening System Product SIRT-Glo™ Control Substrate HeLa Nuclear Extract Nicotinamide Size Cat.# Price (Fr) 10 ml G6450 578.00 Size Conc. 35 µl For Research Use Only. Not for Use in Diagnostic Procedures. Description: The SIRT-Glo™ Assays and Screening Systems are singlereagent-addition, homogeneous, luminescent assays that measure the relative activity of the NAD+-dependent histone deacetylase (HDAC) class III enzymes (sirtuins; SIRTs) from purified enzyme sources. The assays use an acetylated, luminogenic peptide substrate that can be deacetylated by SIRT 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 SIRT-mediated luminescent signal is persistent with a half-life of greater than 3 hours, allowing batch processing of multiwell plates. The SIRT-Glo™ Assay is broadly useful for NAD+-dependent Sirtuin enzymes. Nicotinamide, included in the SIRT-Glo™ Screening Systems or available separately, is a known inhibitor of SIRTs and used as a positive control inhibitor. Nicotinamide is supplied at a concentration of 1M in SIRT-Glo™ Buffer. The HeLa Nuclear Extract, included in the SIRT-Glo™ Screening Systems or available separately, may be used as an assay chemistry control. HeLa Nuclear Extract is supplied at a concentration of 5mg/ml. The SIRT-Glo™ Control Substrate, only available separately, is a non-acetylated form of the SIRT-Glo™ Substrate with the same amino acid sequence and can be used with the SIRT-Glo™ Assays and Screening Systems to confirm true SIRT 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 SIRT-Glo™ Reagent prepared with components in the SIRT-Glo™ Assays or Screening Systems. 10,000 1,000 SIRT1 SIRT2 SIRT3 SIRT4 SIRT5 SIRT6 100 5 × 10 ml G6451 2168.00 100 ml G6452 3468.00 10 ml G6470 615.00 5 × 10 ml G6471 2307.00 Cat.# Price (Fr) G6460 353.00 10 µl 5 mg/ml G6570 165.00 30 µl 1 M G6540 113.00 Features: • Simple Measurement of Deacetylating Activities: Use a singlereagent-addition, homogeneous, add-mix-measure protocol for easy implementation from benchtop to screening. • Highly Sensitive: 10- to 100-fold higher sensitivity than comparable fluorescence methods. • Fast Data Acquisition: Measure maximum signal in as little as 10-15 minutes with persistent glow-type steady-state signal. Storage Conditions: Store the SIRT-Glo™ Assay components and SIRT-Glo™ Control Substrate at –20°C. Store HeLa Nuclear Extract at –70°C. Protocol SIRT-Glo™ Assay and Screening System Technical Manual Part# TM336 3 10 1 110 100 Recombinant SIRT Enzyme (ng/ml) Broad linearity and isoenzyme utility. 1,000 For complete and up-to-date product information visit: www.promega.com/catalog 53 Cell Signaling Signal-to-Noise Ratio 8910MA Pagina 56
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