The most popular applications of GFPs involve exploiting them for imaging, such as localization and dynamics of specific organelles and expression of chimeric or recombinant proteins. However, increased demands for higher-throughput and rapid detection have resulted in methods to exploit their use. Such assays have been adapted to a microplate format and read on fluorescent capable microplate instruments to meet the increased through-put demands. Several commercially available GFP based kits and reagents are available to assist researchers. For example GFP can be stably transfected into a model cell line to act as a gene reporter to quantify activation or inhibition at targets for pharmacological intervention6. Other methods focus on biochemical interactions such as cell signaling by detecting intracellular calcium mobilization7 or generation of hydrogen peroxide8. Several applications have also been developed based on fluorescent resonance energy transfer (FRET) using GFP derived proteins; the ability of one fluorescing compound to transfer, upon excitation, emission energy to excite a second molecule when in close proximity9. The resulting emission intensity of the second or acceptor molecule reflects the proximity of the two molecules. These applications include those where colocalization of two or more proteins can be determined below the theoretical optical resolution of current imaging technology (less than 10 nm). These types of studies are well suited to a microplate format in densities up to 1536-wells/plate. For additional information regarding applications of FRET see An Introduction to Fluorescence Resonance Energy Transfer (FRET) Technology and its Application in Bioscience on the BioTek website10.
The instrumentation itself varies in optical capability; 1) filter based optical systems allow increased light intensity, relative to monochromator based systems, for sample excitation at the filter determined wavelength and bandpass 2) monochromator based systems allow for broad wavelength and bandpass selection for both excitation and emission but exhibit decreased light intensity relative to filter based instruments. Both types of instruments have their positive and negative aspects depending on the nature of the sample and experimental conditions11.
In the future we expect to see GFP used in such applications as identifying cell markers such as Oct4, Sox2 and Nanog in iPS cells in a microplate format to quantify cell induction when exposed to small molecule mimetics to endogenous transcription factors.
If this is an area you are currently working on we would like to hear from you.
As BioTek gears up our new cell culture facility over the next several months are there specific GFP or fluorescent based applications that we might help you optimize on our instruments?
- Prendergast F, Mann K (1978). "Chemical and physical properties of aequorin and the green fluorescent protein isolated from Aequorea forskålea". Biochemistry 17 (17): 3448–53.
- Prasher D, Eckenrode V, Ward W, Prendergast F, Cormier M (1992). "Primary structure of the Aequorea victoria green-fluorescent protein". Gene 111 (2): 229–33.
- "The Nobel Prize in Chemistry 2008". 2008-10-08. Retrieved 2009-10-14.
- Thastrup O, Tullin S, Kongsbak Poulsen L, Bjørn S (1995). "Fluorescent Proteins". US patent. Retrieved 2009-10-14.
- http://www.scholarpedia.org/article/Fluorescent_proteins. Retrieved 2009-10-14.
- http://www.systembio.com/index.php?id=product_25. Retrieved 2009-10-14.
- http://www.enzolifesciences.com/ENZ-51020/gfp-certified-fluoforte-calcium-assay-kit-for-microplates/. Retrieved 2009-10-14.
- Belousov, V.V., Fradkov, A.F., Lukyanov, K.A., Staroverov, D.B., Shakhbazov, K.S., Terskikh, A.V., and Lukyanov, S. (2006) Genetically encoded fluorescent indicator for intracellular hydrogen peroxide, Nat. Methods, 3, 281-286.
- dos Remedios, C.G. and Moens, P.D. (1995) Fluorescence resonance energy transfer spectroscopy is a reliable "ruler" for measuring structural changes in proteins. Dispelling the problem of the unknown orientation factor, J. Struct. Biol., 115, 175-185.
- http://www.biotek.com/resources/articles/fluorescence-resonance-energy-transfer.html. Retrieved 2009-10-14.
- http://americanbiotechnologylaboratory.texterity.com/abl/200608/?pg=18. Retrieved 2009-10-14.
By: BioTek Instruments