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Measuring Intracellular Enhanced Green Fluorescent Protein With the VersaFluor Fluorometer

Contributed by Jeff Wilson, and Andrew Segal, M.D., Genitrix, LLC, Cambridge, MA


Introduction
The Green Fluorescent Protein (GFP) derived from Aqueorius victoria has recently become a popular choice as a reporter gene. GFP can be used, for example, to study promoter function and to track the expression, localization, and function of proteins to which it has been recombinantly fused.1,2 When stimulated by light the molecule fluoresces without addition of any cofactors or substrates. For our studies we have used the Enhanced Green Fluorescent Protein gene (EGFP), a red-shifted variant of the wild-type protein, as a reporter gene in Jurkat cells. EGFP has an excitation maximum at 488 nm, and emission maximum at 507 nm. It is very resistant to photobleaching and is reported to maintain stable fluorescence from pH 7.0 to 11.5.3 In fluorometric assays, purified EGFP can be measured at concentrations in the low nanogram range.

In recent experiments we have shown that intracellular EGFP can be assayed using a VersaFluor fluorometer. Using the EX490/10 and EM510/10 filter set, we compared pEGFP-n2 transfected Jurkat cells with wild-type Jurkat cells. To approximate the amount of EGFP produced by transfected Jurkat cells, a standard curve with recombinant Enhanced Green Fluorescent Protein (rEGFP) was also generated. The methods and results from those experiments are reported here.


Methods
Jurkat cells (clone E6-1 from ATCC), a leukemic human T-cell line, were maintained in RPMI 1640 media with 2 mM L-glutamine and supplemented with 2.5 g/l glucose, 10 mM HEPES, and 1.0 mM sodium pyruvate, in addition to 10% fe
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