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High-throughput analysis of membrane protein structure-function and signal transduction kinetics using robotic fluorescence and luminescence assays

Reference	BB/C511221/1
Principal Investigator / Supervisor	Dr Alan Bateson
Co-Investigators / Co-Supervisors	Professor Stephen Baldwin, Professor David Beech, Professor John Colyer, Dr Hugh Pearson, Professor Chris Peers, Professor Asipu Sivaprasadarao
Institution	University of Leeds
Department	Institute of Membrane & Systems Biology
Funding type	Research
Value (£)	40,063
Status	Completed
Type	Research Grant
Start date	01/02/2004
End date	31/07/2005
Duration	18 months

Abstract

FlexStationII96/384 (Molecular Devices) work station is a bench-top fluorometor with integrated liquid handling for fluorescence or luminescence measurement in 96- or 384-well formats. Millisecond sampling resolution allows analysis of rapid events such as membrane depolarisation, ion-channel activity, Ca2+ mobilisation, 2nd messenger signalling and fast kinetic analysis. Dual monochromators allow multi-wavelength dyes to be used and full optimisation of excitation and emission wavelengths. Most importantly data sets are generated on an experimental timescale that is orders of magnitude quicker than traditional plate reader- or microscope-based assays thereby increasing efficiency. Currently there is no access to such equipment at the University of Leeds. All the proposed projects require high-throughput assay systems of fluorescence or luminescent measurements for determination of intracellular Ca2+ levels, membrane potential, and chemiluminescence. Quantitative, high-throughput BRET and FRET assays are also possible with this equipment. Major projects proposed by the applicants:- AB, Pharmacological and molecular analysis of novel interactions between neuronal ligand-gated ion channels and voltage-dependent calcium channels; DB, Functional testing of putative protein partners of TRPC5 calcium channel; AS, Quantitative measurement of surface expression of ATP-sensitive potassium channels (in normal and disease states) using a chemiluminescence-based assay; SB, High-throughput assays of eukaryote membrane transporter function, JC Rapid kinetic analysis of signal transduction chemistry in living cells; CP & HP, Screening of antioxidant compounds useful in determining the role of reactive oxygen species in oxygen sensing systems. While this application is primarily driven by the co-applicants listed above, a large number of researchers require such equipment, including members of the Centre for Integrative Membrane Biology and CRISTAL. Many of these projects are associated with a major BBSRC-funded initiative at Leeds ¿ the Membrane Protein Structure Initiative (SPORT) and the equipment will be located in the newly refurbished (4.8 million pound) space housing the Centre for Integrative Membrane Biology. Further, it will add significantly to the capacity and diversity of the faculty¿s existing Bioimaging Facility which can provide high resolution data, but cannot make high-throughput measurements.

Summary

unavailable

Committee	Closed Committee - Biomolecular Sciences (BMS)
Research Topics	X – not assigned to a current Research Topic
Research Priority	X – Research Priority information not available
Research Initiative	Research Equipment Initiative 2004 (RE4) [2004]
Funding Scheme	X – not Funded via a specific Funding Scheme

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