User:Laura Carbone/Sandbox 1: Difference between revisions
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While GFP can be incorporated into most prokaryotic systems, expression in eukaryotic systems may be limited to the cytoplasm and the nucleus, as GFP does not penetrate the nucleolus or vesicular organelles. However, highly specific intracellular localization can still be achieved in eukaryotes,<ref name="Yang" /> which can help to avoid the difficulties associated with adding extrinsic dyes.<ref name="Van">van Thor JJ, Sage, JT. 2006. Charge transfer in green fluorescent protein. Photochemical & Photobiological Sciences. 5:597-602. DOI 10.1039/b516525c.</ref> However, this ability to generate fluorescence within live tissues in the absence of cofactors gives GFP the key for use in biological research. After an in-frame fusion to the protein of interest, the resulting chimeric protein can be expressed in a cellular environment to monitor function or activity<ref name="Pollock">[http://www.sciencedirect.com/science?_ob=ArticleURL&_udi=B6TCX-3WRB4G1-5&_user=4187488&_coverDate=02%2F01%2F1999&_rdoc=1&_fmt=high&_orig=search&_sort=d&_docanchor=&view=c&_acct=C000062504&_version=1&_urlVersion=0&_userid=4187488&md5=d50508d087b9a1e500d68a17d2cdb69a], Pollock, BA, and Heim, R. 1999. Using GFP in FRET-based applications. Trends in Cell Biology. 9 (2): 57-60. DOI 10.1016/SO962-8924(98)01434-2.</ref>. | While GFP can be incorporated into most prokaryotic systems, expression in eukaryotic systems may be limited to the cytoplasm and the nucleus, as GFP does not penetrate the nucleolus or vesicular organelles. However, highly specific intracellular localization can still be achieved in eukaryotes,<ref name="Yang" /> which can help to avoid the difficulties associated with adding extrinsic dyes.<ref name="Van">van Thor JJ, Sage, JT. 2006. Charge transfer in green fluorescent protein. Photochemical & Photobiological Sciences. 5:597-602. DOI 10.1039/b516525c.</ref> However, this ability to generate fluorescence within live tissues in the absence of cofactors gives GFP the key for use in biological research. After an in-frame fusion to the protein of interest, the resulting chimeric protein can be expressed in a cellular environment to monitor function or activity<ref name="Pollock">[http://www.sciencedirect.com/science?_ob=ArticleURL&_udi=B6TCX-3WRB4G1-5&_user=4187488&_coverDate=02%2F01%2F1999&_rdoc=1&_fmt=high&_orig=search&_sort=d&_docanchor=&view=c&_acct=C000062504&_version=1&_urlVersion=0&_userid=4187488&md5=d50508d087b9a1e500d68a17d2cdb69a], Pollock, BA, and Heim, R. 1999. Using GFP in FRET-based applications. Trends in Cell Biology. 9 (2): 57-60. DOI 10.1016/SO962-8924(98)01434-2.</ref>. | ||
===Use of GFP as a Fusion Tag=== | |||
One of the most common applications of GFP in research utilizes GFP as a fusion tag to a protein of interest in order to observe the activity of the protein. GFP is fused in frame with the gene encoding the protein of interest, resulting in a chimera that is both functional (hopefully) and fluorescent to be expressed in the organism. This technique has been used successfully in nearly every cell organelle, including the plasma membrane, nucleus, endoplasmic reticulum, Golgi apparatus, secretory vesicles, mitochondria, peroxisomes, vacuoles, and phagosomes. GFP is most commonly fused to either terminal end of the protein gene, although it may be possible to insert it onto a noncritical exterior loop or domain depending on the structure of the protein in question. For example, residues 2-233 of GFP were inserted between the last transmembrane segment and long cytoplasmic tail of a Shaker potassium channel in experiments done by Siegel and Isacoff (1997).<ref name="Tsien" /> | |||
===Use of GFP in FRET=== | ===Use of GFP in FRET=== | ||