User:Laura Carbone/Sandbox 1: Difference between revisions

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===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 as an Active Indicator===

Because the β-can structure protects the chromophore from the surrounding environment, it is difficult to use wild-type GFP as an active indicator of changing conditions. However, environmental indicators have been created by combining various GFP mutants created from random and directed mutagenesis. It is also possible to incorporate phosphorylation sites into the GFP structure such that phosphorylation or dephosphorylation will induce major changes in fluorescence. For example, the Shaker fusion protein mentioned in the section above was the first genetically encoded optical sensor of membrane potential that induced at most a 5% decrease in fluorescence in phosphorylated conditions. However, the most general way to make biochemically sensitive GFPs is to exploit FRET as described in the following sections.<ref name="Tsien" />

===Use of GFP in FRET===

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 ===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 as an Active Indicator===
+Because the β-can structure protects the chromophore from the surrounding environment, it is difficult to use wild-type GFP as an active indicator of changing conditions.  However, environmental indicators have been created by combining various GFP mutants created from random and directed mutagenesis.  It is also possible to incorporate phosphorylation sites into the GFP structure such that phosphorylation or dephosphorylation will induce major changes in fluorescence.  For example, the Shaker fusion protein mentioned in the section above was the first genetically encoded optical sensor of membrane potential that induced at most a 5% decrease in fluorescence in phosphorylated conditions.  However, the most general way to make biochemically sensitive GFPs is to exploit FRET as described in the following sections.<ref name="Tsien" />
 ===Use of GFP in FRET===