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GFP Assays: Live–Cell Translocation Assays
- 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, 10 mg)
- 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.
Technical Specifications
Assay | Signaling Pathways | Application areas | Translocation |
| Stress response pathways | Inflammation, neuronal growth and differentiation, pain, CNS disorders, ischemia, seizures, skeletal muscle regulation | Nucleus to cytoplasm |
| Cytokinesis, phagocytosis, pinocytosis, axon outgrowth, morphogenesis, cell-cell contacts, cell polarity, transformation, adhesion, migration | Colon and breast cancer, cardiac hypertrophy, myofibrillogenesis, pro-inflammatory signaling, leukemia, rheumatoid arthritis, progression to AIDS | Cytoplasm to cell surface ruffles |
| PI(4,5)P2 signaling pathways, adrenoreceptor function, cytoskeletal organization, responses to purinergic agonists, mechanical stress pathways | Cardiac diseases/ hypertrophy, cardiac injury, Alzheimer’s disease, bipolar disorders | Cell surface to cytoplasm |
| Growth factor (RTK) and cytokine signaling pathways, PI(3)P levels (class III PI3Kinase sensor) | Identification of class-specific PI3K inhibitors for development of cancer treatments, immuno-suppressants and anti-inflammatory drugs | Endosomes to cytoplasm |
| Cell survival, proliferation, apoptosis, insulin response pathways | Cancer (breast, prostate, ovarian, gastric), tumor progression, drug and radiation resistance in cancer therapy, invasion, angiogenesis, diabetes | Cytoplasm to cell surface ruffles |
| Acute-phase response pathways, cytokine signaling, leptin signaling | Acute-phase responses, oncogenesis (hematologic, breast, head, neck, prostate cancers), obesity | Cytoplasm to nucleus |
| Immune responses (IL2, IL4, TNFa gene expression), prostaglandin signalling | Oncogenesis, tumor progression, cardiac hypertension, embryonic development, tissue repair, immune function | Cytoplasm to nucleus |
| Cell Cycle Analysis, cell survival, proliferation. | Desired effects: identification of aniti-mitotic or anti-proliferatives compounds
Undesired effects: identification of toxic compounds | Cytoplasm to whole cell to nucleus |
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