Download GFP Assays: Live–Cell Translocation Assays

GFP Assays: Live–Cell Translocation Assays
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Validated stable cell line: start screening immediately without having to spend months
establishing a cell line in-house.
Expression vector: offers the flexibility to work with transients and alternative host cell
lines
Complete right to use: no additional license negotiations are required prior to using the
assay
Utilizes Aequorea victorea GFP: the established benchmark fluorescent protein
technology
Live cellular translocation assays employing GFP are becoming increasingly attractive in
drug discovery for the development of pharmaceutical screens. Previously intractable targets
can be measured by quantitating the real time distribution of GFP-tagged proteins after
treatment with test compounds. Exploiting GFP to its full potential requires access to
advanced live-cell imaging systems and validated assay technologies.
In collaboration with BioImage, Amersham Biosciences has developed a set of translocation
assays. These live-cell assays can be used to track protein movements within intra-cellular
pathways and highlight any effects caused by potential drug candidates. They also allow you
to detect more specific agonists and antagonists and witness that your target protein is active.
The results of a translocation assay will give you the clearest picture yet of the effects of your
compound on the dynamics within a living cell.
The ability to observe the effects on a protein's movement due to your compound (rather than
simply measuring changes in its concentration) represents the opportunity to identify a new
type of pharmaceutical. Translocation assays therefore represent a new direction for drug
discovery.
A GFP translocation assay system provides an extensively validated resource for screening
and profiling drug effects in living cells. Each assay system comprises of the following:
• Fully validated stable cell line (2 vials, 106 cells per vial)
• Expression vector containing cDNA for the GFP fusion protein (1 vial, 10ug)
• Rights to use; covering patents relating to GFP, the CMV promoter and the fusion
protein, if appropriate.
• Full technical support manual (including validation data and protocols).
These assay systems allow focused real time visualization of your drug's interactions with key
signaling pathways without the need for consumable detection reagents. They also provide a
view of drug-target interactions with greater speed and precision than measuring gene
expression or other downstream events.
Ordering information
Description
GFP-MAPKAP-k2 Assay, non-profit research
Code Number
25-9002-60
GFP-Rac1 Assay, Screening
25-9002-61
GFP PLCδ-PH domain Assay Screening
25-9002-62
EGFP-2xFYVE Assay, Screening
25-9002-63
AKT1-EGFP Assay, Screening
25-9002-58
EGFP-STAT3 Assay, Screening
25-9002-64
EGFP-NFAT c1 Assay, Screening
25-9002-65
EGFP-SMAD2 Assay, Screening
25-9002-66
Technical Specifications
Assay
Signalling Pathways
Application areas
Translocation
MAPKAP-k2
Stress response pathways
Nucleus to cytoplasm
RAC1
Cytokinesis, phagocytosis,
pinocytosis, axon outgrowth,
morphogenesis, cell-cell contacts,
cell polarity, transformation,
adhesion, migration
PLCδ-PH domain
PI(4,5)P2 signalling pathways,
adrenoreceptor function,
cytoskeletal organization,
responses to purinergic agonists,
mechanical stress pathways
Growth factor (RTK) and cytokine
signalling pathways, PI(3)P levels
(class III PI3Kinase sensor)
Inflammation, neuronal
growth and differentiation,
pain, CNS disorders,
ischemia, seizures, skeletal
muscle regulation
Colon and breast cancer,
cardiac hypertrophy,
myofibrillogenesis, proinflammatory signalling,
leukemia, rheumatoid
arthritis, progression to AIDS
Cardiac diseases/
hypertrophy, cardiac injury,
Alzheimer’s disease, bipolar
disorders
Identification of classspecific PI3K inhibitors for
development of cancer
treatments, immunosuppressants and antiinflammatory drugs
Cancer (breast, prostate,
ovarian, gastric), tumor
progression, drug and
radiation resistance in
cancer therapy, invasion,
angiogenesis, diabetes
Acute-phase responses,
oncogenesis (hematologic,
breast, head, neck, prostate
cancers), obesity
Immunosuppression,
muscle growth
Endosomes to cytoplasm
Oncogenesis, tumor
progression, cardiac
hypertension, embryonic
development, tissue repair,
immune function
Cytoplasm to nucleus
FYVE
AKT1
Cell survival, proliferation,
apoptosis, insulin response
pathways
STAT3
Acute-phase response pathways,
cytokine signalling, leptin
signalling
NFAT c1
Immune responses (IL2, IL4, TNFa
gene expression), prostaglandin
signalling
TGF-beta signalling, growth,
differentiation, cell survival,
regulation of cell proliferation and
migration, elaboration of
extracellular matrix
SMAD2
Cytoplasm to cell surface
ruffles
Cell surface to cytoplasm
Cytoplasm to cell surface
ruffles
Cytoplasm to nucleus
Cytoplasm to nucleus
Algorithm Compatibility
Assay
Translocation
Analysis Module
MAPKAP-k2
Nucleus to cytoplasm
Nuclear Trafficking
RAC1
Cytoplasm to cell surface
ruffles
Cell surface to cytoplasm
Plasma Membrane Spot
PLCδ-PH
domain
FYVE
AKT1
Endosomes to cytoplasm
Plasma Membrane
Trafficking
Granularity
IN Cell Analyzer
1000
Available
IN Cell Analyzer
3000
Available
TBD
Available
Q4 2003
Available
Available
Available
TBD
Available
Plasma Membrane Spot
STAT3
Cytoplasm to cell surface
ruffles
Cytoplasm to nucleus
Nuclear Trafficking
Available
Available
NFAT c1
Cytoplasm to nucleus
Nuclear Trafficking
Available
Available
SMAD2
Cytoplasm to nucleus
Nuclear Trafficking
Available
Available
G2M CCPM
Cell Cycle
Cell Cycle Trafficking
Q4 2003
Available
Cell Cycle: G2M Cell Cycle Phase Marker
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Dynamic, non-toxic reporter of cell cycle status in living cells.
Enables examination of cell cycle over extended time periods.
Higher sample throughput on the IN Cell Analyzer 3000 and IN Cell Analyzer 1000 than
flow cytometry.
Used in conjunction with the Cell Cycle Trafficking analysis module it is possible to
distinguish four stages of the cell cycle: G1/S, G2, prophase and the other stages of
mitosis.
The assay depends on controlling the expression levels and location of GFP in a cell cycle
dependent manner. This is achieved by using functional regions from cell cycle proteins that
control reporter expression, destruction and translocation from the cytoplasm to the nucleus.
The G2/M cell cycle phase marker assay uses cell cycle specific promoter, destruction and
localisation sequences from the key cell cycle modulating protein cyclin B1 fused to GFP.
The expression of GFP is restricted to late S and G2 phases by the cyclin B1 promoter. It
translocates from the cytoplasm to the nucleus at prophase due to the cyclin B1 cytoplasmic
retention sequence (CRS) and is specifically degraded in metaphase due to the presence of the
cyclin B1 D-box . Cell cycle status is therefore determined by interrogating the position and
level of GFP in a cell. The assay has been designed for screening of cell cycle perturbing
drugs, toxicology testing and lead profiling.
Ordering information
Description
G2M Cell Cycle Phase Marker Assay, Non profit research
Code Number
25-9002-55