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GFP-Rac1
The GFP-Rac1Assay follows the redistribution of GFP-Rac1 in response to growth and motility promoters. It monitors the redistribution of activated Rac1 from the cytoplasm to the membrane ruffles that are formed upon the reorganization of the actin filaments in the plasma membrane. Rac1 is a regulator of the assembly and organization of the actin cytoskeleton and is therefore critical to many physiological processes including the control of cell shape, cell motility, cell polarity and adhesion cytokinesis, and other related processes. The assay consists of an extensively validated stably transfected cell line and expression vector for use with automated and traditional confocal and epifluorescent 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 licenses are required prior to using the assay
Move towards a greater understanding>
A measured response>
Results with a range of imaging platforms>
Assay components>
Move towards a greater understanding
| Unstimulated cell | Stimulated cell |
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Fig 1. Schematic of assay principle.
- Direct quantitation of agonist-induced Rac1 movement and location: makes it possible to screen for compounds that have a positive or negative effect on the cascade that leads to the activation of Rac1
- Utilizes Aequorea victorea GFP: the established benchmark fluorescent protein technology
Rac1 induces the formation of web-like cellular extensions (lamellipodia) and is a GTP-binding protein from the Rho-family of proteins. Rho-family members regulate a variety of cellular processes such as in cytokinesis, phagocytosis, pinocytosis, axon outgrowth, morphogenesis, cadherin-derived cell-cell contacts, cell polarity, cell transformation, cell-substratum contacts, and cell migration. Cell migration is crucial for embryonic development, the inflammatory immune response, wound repair, tumor formation, and metastasis. All of these processes are controlled primarily through the assembly and organization of the actin cytoskeleton.
Rac1, Cdc42, and RhoA are the most extensively characterized Rho-family members and together they serve as molecular switches in regulating a wide range of essential biochemical pathways in eukaryotic cells. Rho-GTPases interact with various subsets of effectors depending upon the cellular context and possibly upon the mechanism of activation. Currently there are three Rac-isoforms reported: Rac1, Rac2, and Rac3.
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A measured response
 | Redistribution of activated Rac1 is measured using an analysis module that quantitates and detects the formation of fluorescent ruffles at the cell surface. |
Fig 2. Insulin dose response curve, data were collected 7 min after addition of the agonist and demonstrate an EC50 = 0.6 nM, (determined on IN Cell Analyzer 3000 using the Plasma Membrane Spot Analysis Module). Error bars indicate SD, n = 6 per data point.
Fig 3. Wortmannin inhibition curve, data were collected 6-8 min after addition of 100-nM Insulin and demonstrate an IC50 = 6.53 nM (determined on IN Cell Analyzer 3000 using the Plasma Membrane Spot Analysis Module). Error bars indicate SD, n = 3 per data point.
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Results with a range of imaging platforms
GFP-Rac1 assay was designed as a live- or fixed-cell system using image acquisition and analysis on the IN Cell Analyzer 3000. 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
- GFP-Rac1 stably expressing cell line, CHO derived, 2 vials, each containing 1 x 106 cells
- pCORON 1000-GFP-Rac1 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 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 AKT1-EGFP 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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