Cell Signaling 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. Description: The GloResponse™ Luciferase Reporter Cell Lines contain optimized, state-of-the-art luciferase reporter technology integrated into a cell line. This allows the rapid development of a reporter assay based on the pathway of interest regulating the luciferase gene. Assays configured using the GloResponse™ Cell Lines are amenable for high-throughput screening. These assays typically have greater response dynamics (fold of induction) than other assay formats and good quality as indicated by the high Z´ values. GloResponse™ Cell Lines were developed to study a variety of signaling pathways. Activators of these pathways may be native to the HEK293 cell line. Activity of non-native activators can be studied after they have been introduced by transfection. GPCRs regulate a wide-range of biological functions and are one of the most important target classes for drug discovery. GPCR signaling pathways can be categorized into three classes based on the G protein α-subunit involved: Gs, Gi/o and Gq. The GloResponse™ CRE-luc2P HEK293 Cell Line can be used to study and configure screening assays for Gs- and Gi/o-coupled GPCRs, which signal through cAMP and the cAMP Response Element (CRE). For Gq-coupled GPCRs, which signal through calcium ion release and activate the Nuclear Factor of Activated T-Cells response element (NFAT-RE), the GloResponse™ NFAT-RE-luc2P HEK293 Cell Line should be used. NF-κB-REs are the DNA binding sequences for the NF-κB transcription factor complex, which is responsible for regulating inflammation, immune response, cell growth and apoptosis. The GloResponse™ NF-κB-RE-luc2P HEK293 Cell Line is designed for rapid and convenient analysis of any cellular response that results in modulation of NF-κB activities. The GloResponse™ 9XGAL4UAS-luc2P HEK293 Cell Line contains nine repeats of GAL4 UAS (Upstream Activator Sequence) driving the transcription of the luciferase reporter gene luc2P in response to binding of a fusion protein containing the GAL4 DNA Binding Domain, such as the Estrogen Receptor Ligand Binding Domain in pBIND-ERα Vector (Cat.# E1390) when activated by a ligand. This makes the cell line suitable for the study of nuclear receptors or can be used to study other types of protein:protein and protein:DNA interactions. The GAL4 DNA Binding Domain partner must be introduced to this cell line by transfection or other similar techniques. The GloResponse™ Cell Lines were generated by clonal selection of HEK293 cells stably transfected with pGL4-based vectors carrying specific response elements for the pathway of interest. These cell lines incorporate the improvements developed for the pGL4 family of reporter vectors for enhanced performance. The destabilized luc2P luciferase reporter is used for improved responsiveness to transcriptional dynamics. The luc2P gene is codon optimized for enhanced expression in mammalian cells, and the pGL4 plasmid backbone was engineered to reduce background reporter expression. The result is a cell line with very high induction levels when the pathway of interest is activated. Features: • Convenient: Prebuilt, optimized luciferase reporter cell lines. • Robust: Large assay window provided by high levels of induction and low background expression. • Faster Results: Improved responsiveness to transcriptional dynamics with destabilized luciferase. Storage Conditions: Place frozen cells in storage at less than or equal to –140°C (mechanical deep freeze or vapor phase liquid nitrogen) until you are ready to thaw and propagate them. We strongly recommend that the cells are propagated, using the provided procedure, as soon as possible. This will ensure the optimal cell viability and assay performance. Protocol GloResponse™ CRE-luc2P HEK293 Cell Line Technical Bulletin GloResponse™ 9XGAL4UAS-luc2P HEK293 Cell Line Technical Bulletin pGL4-RE-luc2P RE pRluc-Neor-GPCR PCMV Part# TB362 GloResponse™ NFAT-RE-luc2P HEK293 Cell Line Technical Bulletin TB363 GloResponse™ NF-κB-RE-luc2P HEK293 Cell Line Technical Bulletin TB380 TB552 15 luc2P PSV40 Hygr GPCR PSV40 Rluc-Neor 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 2 × 105 1 × 105 0 pergolide apomorphine SKF38393 dopamine dihydrexidine Two plasmids involved in the dual-luciferase GPCR assay. RE, response element/promoter; luc2P, destabilized firefly luciferase with PEST sequence; –9 –8 –7 log [agonist], M 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). –6 –5 For complete and up-to-date product information visit: www.promega.com/catalog 277 Reporter Assays and Transfection Luminescence (RLU) 6632MB 5251MA Pagina 280
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