Aoki Kazuhiro, Komatsu Naoki , Goto Akihiro, and Matsuda Michiyuki
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.
Small GTPases act as molecular switches that regulate a variety of cellular functions, such as proliferation, cell movement and vesicle trafficking. Genetically encoded biosensors based on the principle of fluorescence resonance energy transfer (FRET) can visualize a spatio-temporal activity of small GTPases in living cells, thereby helping us to understand the role of small GTPases intuitively and vividly. Here we describe protocols of live cell imaging with the FRET biosensors. There are several types of FRET biosensors; this protocol focuses on intramolecular or unimolecular FRET biosensors of small GTPases that are made up of donor and acceptor fluorescence proteins, a small GTPase, its binding partner, and, if necessary, a subcellular localization signal. These FRET biosensors uncover the spatio-temporal activity of the small GTPases in living cells, which could not be obtained by conventional biochemical methods. Preparation of FRET biosensors and cell culture takes 6 d. Imaging and processing take 3-4 d to complete.
… “ LED illumination system, CoolLED precisExcite (Molecular Device) or 75-W Xenon arc lamp and the power supply (Olympus)”…
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pE-100: A range of compact, simple-to-use, single wavelength illumination systems for screening fluorescence.
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