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Measurement of Green Fluorescent Protein in the SpectraMax Gemini XS Spectrofluorometer (MaxLine Application Note #44)

Simon Lydford1 and Thomas Giller2
1
Molecular Devices Limited and 2Axovan Ltd.


INTRODUCTION
GFP (Green Fluorescent Protein) is an extremely stable protein of 238 amino acids produced by the jellyfish Aequorea victoria1 that fluoresces in the lower green region of the visible spectrum. Over the past few years GFP has become one of the most widely used tools in molecular and cell biology as the GFP gene can be expressed in non-homologous species. Additionally, the fact that GFP can generate a highly visible fluorescence that requires no additional substrates has made it valuable as a marker for gene expression, as a cell lineage tracer or as an in vivo covalently attached fluorescent label replacing more invasive and unwieldy fluorescent staining techniques. GFP can be monitored non-invasively and may be used as a quantitative marker in living cells. GFP can also be used as a tag for studying real time localization and movement of protein-GFP chimeras within cells2.

However, there have been some technical difficulties associated with the use of GFP. One major concern is the excitation and emission spectra of the protein. Wild type GFP has two excitation peaks, a major one at 395 nm (in the long UV range) and a smaller one at 475 nm (blue) with an emission peak at 509 nm (green). For wild type GFP, it has been found that exciting the protein at 395 nm causes rapid quenching of the fluorescence. Recently, some modified forms of the GFP gene have been produced with different spectral properties. For example, the major excitation peak has been red-shifted to 490 nm and the emission peak staying at 509 nm. Perhaps more importantly, these modifications generally increase the fluorescent output3.

As the GFP signal can be weak and hard to detect, especially when expressed at low levels, fluorescent cells are usually observed using
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