Abstract
The cloning of green fluorescent protein (GFP) has opened a new arena of protein labeling and has become a new alternative to existing markers or dyes. Main advantages are its stability and its fluorescent activity independent on the binding of other ligands or proteins. The use of GFP-tagged proteins has found multiple applications in molecular biology such as lineage tracing assays, cell fusion assessment, and/or immunodetection, among others. High-resolution imaging combined with immunogold labeling for GFP provides the best correlation with subcellular localization, and it allows detection of membrane-bound GFP-tagged receptors and cytosolic and/or nuclear proteins. In this chapter, we attempt to summarize the state-of-the-art in GFP detection, current protocols, advantages, and pitfalls, and we describe a method, used in our laboratory, which combines confocal fluorescence microscopy with transmission electron microscopy (TEM) for the study of cell cultures at the ultrastructural level.
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Acknowledgments
We thank M. Salomé Sirerol-Piquer, Clara Alfaro-Cervelló, Ulises Gómez-Pinedo, and Mario Soriano-Navarro for their useful contribution. We thank Dr. Engelhardt for kindly provide mGFP transgenic mice, and Maria Duran-Moreno for sharing her GFP picture. This work was supported by Spanish MINECO grants (Instituto Salud Carlos III-RETIC TerCel and SAF2012-33683, to JMGV) and pre- and postdoctoral fellowships (AP2010-4264 and CM12/00014 to ACS and SGP, respectively), the University of Valencia predoctoral fellowship (to PGB), and Generalitat Valenciana predoctoral fellowship Santiago Grisolia (to MF).
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Gil-Perotin, S. et al. (2016). Localization of GFP-Tagged Proteins at the Electron Microscope. In: Luján, R., Ciruela, F. (eds) Receptor and Ion Channel Detection in the Brain. Neuromethods, vol 110. Humana Press, New York, NY. https://doi.org/10.1007/978-1-4939-3064-7_14
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DOI: https://doi.org/10.1007/978-1-4939-3064-7_14
Publisher Name: Humana Press, New York, NY
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