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Protein-protein interactions within the apoptosis regulating Bcl-2 family by fluorescence resonance energy transfer and flow cytometry
Reference
E06476
Principal Investigator / Supervisor
Professor Caroline Dive
Co-Investigators /
Co-Supervisors
Professor John Hickman
Institution
The University of Manchester
Department
Life Sciences
Funding type
Research
Value (£)
177,016
Status
Completed
Type
Research Grant
Start date
14/10/1996
End date
13/10/1999
Duration
36 months
Abstract
An assay of protein-protein interactions in intact cells will be developed using flow cytometry. Bcl-2, it's homologues (e.g. Bax) and accessory proteins (e.g. Bag- 1) form homo and heterodimers which regulate apoptosis. Fluorescence Resonance Energy Transfer (FRET) will be measured between antibodies to e.g. Bcl-2 and Bax tagged with fluorescein or coumarin as the acceptor-donor pair. Coumarin (Bax) will be excited at 356nm resulting in blue fluorescence; binding of Bax to Bcl-2 (FITC) will result in FRET derived green fluorescence. The ratio of blue/green fluorescence will indicate differences in protein-protein binding. The assay will be validated by expression of mutant proteins that no longer interact. The ability to measure protein- protein interactions in intact cells, and correlation of this with cell fate will advance understanding of the regulation of apoptosis.
Summary
unavailable
Committee
Closed Committee - Engineering & Biological Systems (EBS)
Research Topics
X – not assigned to a current Research Topic
Research Priority
X – Research Priority information not available
Research Initiative
X - not in an Initiative
Funding Scheme
X – not Funded via a specific Funding Scheme
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