GFP-PLCd-PH domain
The GFP-PLCd-PH Domain Assay provides a live-cell screening resource for inducible regulation of PLCd- and PI(4,5)P2-mediated processes. Cleavage of phosphoinositol lipids in the cell membrane constitutes a key event in signal transduction. The PH domain of PLCd associates with specific phosphoinositides at the inner surface of the plasma membrane in resting cells, and translocates transiently into the cytoplasmic space upon pathway stimulation. 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 license negotiations 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
 | Agonist
20-25 s | | 25-120 s | |
 |
Unstimulated cell:
GFP-PLCd-PH domain is most
concentrated at the plasma
membrane | | Stimulated cell:
GFP-PLCd-PH domain
translocates transiently
to cytoplasmic space | | Post stimulated cell:
GFP-PLCd-PH domain
reaccumulates at the
plasma membrane |
Fig 1. Schematic of assay principle.
- Direct quantitation of the transient intracellular translocation of PLCd-PH domain: makes it possible to screen for compounds that induce this rapid event
- Utilizes Aequorea victorea GFP: the established benchmark fluorescent protein technology
PLCd is expressed in a variety of tissues, and normally resides at the plasma membrane by virtue of its PH domain, which binds specific phophoinositides and inositol phosphates, particularly phosphoinositol 4,5-bisphosphate (PI(4,5)P2). Dynamic and tightly regulated changes in PI(4,5)P2 at the plasma membrane play critical roles in key cellular processes such as endocytosis, phagocytosis, regulation of the actin cytoskeleton, and activation of PLCd and its downstream signaling pathways A major function of the PH domain of PLCd is to modulate enzyme activity that leads to generation of second messengers such as IP3 and DAG in response to activation of receptors by hormones, neurotransmitters, growth factors, and other molecules. Deregulation of PLCd has been linked to diseases such as Alzheimer's and in cancers including intestinal metaplasia and adenocarcinoma.
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A measured response
The degree of translocation of the GFP-PLCd-PH domain fusion protein from the cellular plasma membrane to the cytoplasm is determined using the Plasma Membrane Trafficking Analysis Module. This module is designed to examine the radial distribution of GFP-PLCd-PH domain fusion protein in a cell. The distribution is investigated by extending defined rays from the seed point origin (nucleus) outwards and towards the periphery of the cell. The intensity of each pixel along the ray is determined and the brightest point (peak) located.
 | The raw peak intensity, distance (intensity weighted or absolute) from the seed point, or the ratio of the peak intensity to other portions of the ray are then examined. The Plasma Membrane Trafficking Analysis Module is kinetic, meaning that the result for each well reflects a percentage change in any value from a pre-stimulation measurement to apost-stimulation measurement. Measuring a change in value instead of absolute values has the advantage of minimizing the variability of starting conditions for the assay. The module also reports the static (pre- or post-stimulation) values. |
Fig 2. ATP dose response curve. Data were collected 25 s after addition of the agonist and demonstrate an EC50 = 5.2 µM (determined on IN Cell Analyzer 3000 using the Plasma Membrane Trafficking Analysis Module). Error bars indicate SD, n = 6 per data point.
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Results with a range of imaging platforms
The GFP-PLCd-PH Domain Assay has been developed as a live-cell system using image acquisition and analysis on the IN Cell Analyzer 3000. The redistribution of PLCd-PH domain is a very rapid and transient response, which is not particularly amenable to fixation. Alternatively, the assay may be used in conjunction with fluorescence microscopes or other automated subcellular imaging platforms that have appropriate analysis software.
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Assay components
- GFP-PLCd-PH Domain stably expressing cell line, CHO derived, 2 vials, each containing 1 x 106 cells
- pCORON 1000-GFP-PLCd-PH Domain 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.
Amersham and Amersham Biosciences are trademarks of Amersham plc. The product is developed and sold under license from: BioImage A/S: Under patents US 6 172 188, EP 851874, EP 986753, and US651802 and other pending and foreign patent applications. Vertex Pharmaceuticals (formerly 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. 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. Columbia University: This product is sold under license from Columbia University under US patent Nos. 5 491 084 and 6 146 826. 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. 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 Dissorders' issued 25 April 2000 and assigned to Sugen Inc, CA, USA. © Amersham Biosciences UK Limited, 2003 - All rights reserved. All goods and services are sold subject to the terms and conditions of sale of the company within the Amersham Biosciences group that supplies them. A copy of these terms and conditions is available on request. 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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