EGFP-2×FYVE
The EGFP-2×FYVE Assay may be used to detect intracellular phosphoinositol 3-phosphate (PI[3]P) levels or as a class III phosphatidyl inositol 3-kinase (PI3-kinase) cellular sensor. The majority of cellular PI(3)P is thought to be generated by the constitutive action of a class III PI3-kinase. Because PI3-kinases are involved in diverse critical signaling and trafficking pathways, PI3-kinase sensors such as EGFP-2×FYVE domain may be useful in the discovery and development of more selective therapeutic PI3-kinase inhibitors. The assay consists of an extensively validated stably transfected cell line and expression vector for use with automated and traditional confocal and epifluorescence microscopes.
- 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
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A measured response >
Results with a range of imaging platforms>
Assay components>
Move towards a greater understanding
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Untreated cell:
EGFP-2×FYVE is concentrated in endosomes | | Treated cell:
EGFP-2xFYVE is redistributed to the cytoplasm |
- Class III PI3 kinase sensor: Direct quantitation of FYVE redistribution from its initial location bound to PI(3)P in early endosomes to the cytoplasm enables intra-cellular detection of class III PI3-kinase activity
- Intracellular PI(3)P monitor: In cells challenged with test compounds that deplete intracellular PI(3)P, EGFP-2x~FYVE bound to PI(3)P on early endosome membranes redistributes from its original position to the cytoplasm
- Pathway profiling: Can be combined with other GFP assays to profile signaling pathways
The discovery of FYVE domains enabled the development of a specific, intra-cellular probe for either PI3-kinase activity or the presence of PI(3)P. The FYVE domain was named after four proteins in which it was originally found: Fab1,YOTB,Vac1p, and EEA1. FYVE domains have eight conserved cysteine residues that coordinate two Zn2+ ions in a specific conformation. The third cysteine residue lies within a highly conserved basic motif:
R(R/K)HHCRxCG, which mediates binding of the inositol head group of PI(3)P. The highly conserved nature of FYVE fingers strongly suggests that they all bind to PI(3)P or a similar ligand.
High-affinity binding of FYVE domains to PI(3)P is thought to depend not only on the inositol head group itself, but also on the presence of intact lipid within a membranous structure. FYVE finger proteins can be subdivided into distinct groups comprising proteins with diverse structures and functions. FYVE domains are shown in live cells to localize to PI(3)P on early endosome membranes in a class III PI3-kinase-dependent fashion.
The EGFP-2xFYVE Assay may therefore be used in conjunction with other phosphoinositide reporters as a class III-specific PI3-kinase sensor or to monitor the presence of PI(3)P. PI3-kinases play critical roles in the regulation of many cellular processes including cell proliferation, survival, motility, and metabolism. They are therefore of great interest as therapeutic targets.
Although developed for use in screening, the EGFP-2xFYVE domain reporter can also be used in conjunction with other GFP assays to profile signaling pathways. For example, the EGFP-2xFYVE and EGFP-SMAD2 assays could be combined in profiling TGFa signaling.
This is because correct localization and function of SMAD2 would be disrupted by compounds that also disrupt FYVE-domain localization. The assay may also be used in conjunction with the AKT1-EGFP Assay and the GFP-PLCd-PH domain Assay to study protein localization domains.
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A measured response
 | The degree of EGFP-2×FYVE localization on early endosomes is measured using the Granularity Analysis Module, which identifies granular fluorescence. Early endosomes are defined as focal regions within the cell having a defined intensity difference from their background. |
Fig 2.Wortmannin dose response curve, data was collected 30 min after addition of Wortmannin. The calculated EC50 was 1.88 nM. Error = ±SD, n = 8 replicates per data point.
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Results with a range of imaging platforms
EGFP-2×FYVE Assay was optimized for image acquisition and analysis on the IN Cell Analyzer 3000 and IN Cell Analyzer 1000 using the Granularity Analysis Module. Alternatively, the assay may be used in conjunction with other automated subcellular imaging platforms that have appropriate analysis software, or fluorescence microscopes.
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Assay components
- EGFP-2×FYVE stably expressing cell line, U-2 OS derived, 2 vials, each containing 1 x 106 cells
- pCORON 1000-EGFP-2×FYVE expression vector 1 vial, containing 10-µg DNA
- User manual detailing full assay protocols and validation data
- Rights to use; covering patents relating to GFP, FYVE domain, and the CMV promoter
* These codes give the user the rights to evaluate the assay for a fixed time period of either 6 or 12 months prior to a full purchase.
Use of the EGFP-2xFYVE Assay is limited as stated in the terms and conditions of sale.
These vary in accordance with the product code purchased.
EGFP = enhanced green fluorescent protein.
This product can be vsiualised on the IN Cell Analysis System - a total solution for high-content sub-cellular analysis in research and drug discovery.
Product is developed and sold under license from: BioImage A/S under patents US 6 172 188, EP 851874 and EP 0986753 and other pending and foreign patent applications. Invitrogen IP Holdings Inc (formerly Vertex Pharmaceuticals and Aurora Biosciences Corporation) under US patents 5 625 048, 5 777 079, 5 804 387, 5 968 738, 5 994 077, 6 054 321, 6 066 476, 6 077 707, 6 090 919, 6 124 128, 6 172 188, European patent 1104769 and Japanese patent JP3283523 and other pending and foreign patent applications. Columbia University under US patent Nos. 5 491 084 and 6 146 826. University of Florida Research Foundation under patents US patents 5,968,750, 5,874,304, 5,795,737, 6,020,192 and other pending and foreign patent applications; and Iowa Research Foundation. The CMV promoter is covered under US patents 5 168 062 and 5 385 839 and its use is permitted for research purposes only. Any other use of the CMV promoter requires a license from the University of Iowa Research Foundation 214 Technology Innovation Center Iowa City IA52242 USA. For customers wishing to use the assay for screening for potential therapeutic agents, attention is drawn to the existence of US Patent Number 6 054 280 'Methods for Diagnosis and Treatment of PH Domain Signal Transduction Disorders' issued 25 April 2000 and assigned to Sugen Inc., CA, USA. Rights to use this product, as configured, are limited to internal use for screening, development and discovery of therapeutic products; NOT FOR DIAGNOSTIC USE OR THERAPEUTIC USE IN HUMANS OR ANIMALS. No other rights are conveyed. Amersham Biosciences UK Limited, Amersham Place, Little Chalfont, Buckinghamshire, England HP7 9NA. Amersham Biosciences AB, SE-751 84 Uppsala, Sweden. Amersham Biosciences Corp, 800 Centennial Avenue, PO Box 1327, Piscataway NJ 08855, USA. Amersham Biosciences Europe GmbH, Munzinger Strasse 9, D-79111, Freiburg, Germany
 | This assay has been developed in collaboration with BioImage.
BioImage is a Danish Biotech company specializing in developing drug
candidates that exert their activity through modulation of protein
translocation. |
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