Science Catalog 2012 Life Cell Signaling PDE-Glo™ Phosphodiesterase Assay Product PDE-Glo™ Phosphodiesterase Assay For Research Use Only. Not for Use in Diagnostic Procedures. Description: The PDE-Glo™ Phosphodiesterase Assay is a luminescent, high-throughput screening (HTS) method for measuring cyclic nucleotide phosphodiesterase activity from purified sources. Cyclic nucleotide phosphodiesterases (PDEs) are involved in a myriad of cellular processes due to their ability to hydrolyze, and thus control, the levels of the second-messenger signaling molecules cAMP and cGMP. The availability of selective inhibitors for PDEs has facilitated their use as tools to study cyclic nucleotide signaling and paved the way to investigate the role of PDEs in cellular and tissue pathologies. The PDE-Glo™ Phosphodiesterase Assay allows lead candidates to be identified from compound libraries. The assay is designed for 384-well plates, but assay volumes can easily be scaled for 96- or 1536-well plates. The PDE-Glo™ Phosphodiesterase Assay is optimized to work with both cAMP- and cGMP-dependent phosphodiesterases. The total time required for the assay from start to finish is less than 1 hour after the PDE reaction is complete. Features: Versatile: Works with both cAMP and cGMP PDEs. Sensitive: • Excellent signal:background ratios. • Scalable to 1536-well plate formats. Fast and Easy to Use: • Assay can be completed in <1 hour. • Homogeneous. Proven Luminescent Technology: • Powered by Ultra-Glo™ Luciferase. • Nonradioactive. No Interference by Fluorescent Compounds. Storage Conditions: Store the system at –20°C. See the product label for the expiration date. Protocol PDE-Glo™ Phosphodiesterase Assay Technical Bulletin PDE cNMP Phosphorylated protein kinase A substrate ADP P cNMP RR C C ATP luciferin + O2 luciferase Active protein kinase A Protein kinase A substrate oxyluciferin + AMP+ Light cNMP = cAMP or cGMP NMP = AMP or GMP The PDE-Glo™ Phosphodiesterase Assay. cNMP RR CC Inactive protein kinase A holoenzyme NMP Ranking compound potency and detection of DRD1 partial agonists. A GloResponse™ CRE-luc2P clone stably expressing dopamine receptor D1 was plated at 10,000 cells/well in a 96-well plate. Each agonist was serially diluted 1:2, then added to wells in replicates of four, beginning with 50μM. Cells were incubated with agonist for four hours, harvested and analyzed using the Dual-Glo™ Luciferase Assay System (Cat.# E2920). Luciferase activity was measured on the GloMax® 96 Microplate Luminometer (Cat.# E6501). 2 × 105 Part# TB353 1 × 105 0 Size Cat.# Price (Fr) 1,000 assays V1361 761.00 10,000 assays V1362 Pls. Enq. GloResponse™ Luciferase Reporter Cell Lines Product GloResponse™ CRE-luc2P HEK293 Cell Line GloResponse™ NFAT-RE-luc2P HEK293 Cell Line Size Cat.# Price (Fr) 2 vials E8500 6721.00 2 vials E8510 6721.00 GloResponse™ NF-κB-RE-luc2P HEK293 Cell Line 2 vials E8520 6721.00 GloResponse™ 9XGAL4UAS-luc2P HEK293 Cell Line 2 vials E8530 6721.00 For Research Use Only. Not for Use in Diagnostic Procedures. For additional information see page XX. pGL4-RE-luc2P RE pRluc-Neor-GPCR PCMV luc2P PSV40 Hygr GPCR PSV40 Rluc-Neor PEST sequence; PSV40, SV40 promoter; Hygr, hygromycin resistance gene; PCMV, CMV promoter; Rluc-neor, Renilla luciferase and neomycin resistance gene fusion. PEST sequences are associated with rapidly degraded proteins. 3 × 105 pergolide apomorphine SKF38393 dopamine dihydrexidine Two plasmids involved in the dual-luciferase GPCR assay. RE, response element/promoter; luc2P, destabilized firefly luciferase with –9 –8 –7 log [agonist], M –6 –5 50 For complete and up-to-date product information visit: www.promega.com/catalog 6289MA Luminescence (RLU) 6632MB 5251MA Pagina 53
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