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Visualization of small GTPase activity with fluorescence resonance energy transfer-based biosensors.

Authors Aoki, K., & Matsuda, M.
Affiliations Laboratory of Bio-imaging and Cell Signalling, Graduate School of Bio-studies, Kyoto University, Kyoto, Japan. Department of Pathology and Biology of Diseases, Graduate School of Medicine, Kyoto University, Kyoto, Japan.
Application Area Science Technology
Abstract Protein kinases are key modulators of intracellular signal transduction cascades, which determine various events in neuronal cells such as replication and differentiation. For many years, protein kinases were analysed mostly by biochemical methods, which could handle the cells only en masse. For a better under- standing of the role of kinases in neuronal cells, one would like to know the subcellular distribution of kinase activities and to follow a particular kinase activity for a specific period in a single cell. Genetically encoded biosensors based on the principle of Förster (or fluorescence) resonance energy transfer (FRET) and fluorescent proteins have been developed to accommodate such requirements. The method involves expression of the FRET biosensors in neuronal cells, time-lapse imaging under fluorescence microscopes, image processing, and quantification of FRET. This technique could be applicable to living organisms ranging from Caenorhabditis elegans to mouse, permitting visualization of spatio-temporal regulation of kinase activities and systemic understanding of the signalling networks in living animals.
Extract … “– LED illumination system, CoolLED precisExcite (Molecular device)”…
Product Associated Features pE-100: A range of compact, simple-to-use, single wavelength illumination systems for screening fluorescence.
Diascopic Technique Bright-Field
Live Cell Issues Photobleaching
Product Type pE-100
Journal Nature protocols
Year of Publication 2009
Country of Publication Japan

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