2013 Promega catalogue

Life
Science
Catalog
2013
Worldwide Contact List
Section
Contents
Cell Signaling
GloResponse Luciferase Reporter Cell
Lines
Product Size Cat.# Price ($)
GloResponse CRE-luc2P HEK293 Cell Line 2 vials E8500 Pls. Enq.
GloResponse NFAT-RE-luc2P HEK293 Cell Line 2 vials E8510 Pls. Enq.
GloResponse NF-κB-RE-luc2P HEK293 Cell Line 2 vials E8520 Pls. Enq.
GloResponse 9XGAL4UAS-luc2P HEK293 Cell Line 2 vials E8530 Pls. Enq.
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: G s ,
G i/o and G q . The GloResponse CRE-luc2P HEK293 Cell Line can be used to
study and configure screening assays for G s - and G i/o -coupled GPCRs, which
signal through cAMP and the cAMP Response Element (CRE). For G q -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 NFAT-RE-luc2P HEK293 Cell Line Technical Bulletin
GloResponse NF-κB-RE-luc2P HEK293 Cell Line Technical
Bulletin
GloResponse 9XGAL4UAS-luc2P HEK293 Cell Line Technical
Bulletin
pGL4-RE-luc2P
RE luc2P PSV40 Hyg r
pRluc-Neo r -GPCR
PCMV GPCR PSV40 Rluc-Neo r
Part#
TB362
TB363
TB380
TB552
Two plasmids involved in the dual-luciferase GPCR assay. RE,
response element/promoter; luc2P, destabilized firefly luciferase with PEST
sequence; P SV40 , SV40 promoter; Hyg r , hygromycin resistance gene; P CMV ,
CMV promoter; Rluc-neo r , Renilla luciferase and neomycin resistance gene
fusion. PEST sequences are associated with rapidly degraded proteins.
Luminescence (RLU)
pergolide
apomorphine
SKF38393
3 × 10 5 dopamine
dihydrexidine
2 × 10 5
1 × 10 5
0
–9 –8 –7 –6 –5
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).
6632MB
5251MA
Table of
Contents
314
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