Abstract: gfp is a natural fluorescent protein derived from the jellyfish aequorea victoria. The mutagenesis of gfp has produced many variants with different spectral properties, which are used both as donors and acceptors of fluorescent resonance energy transfer. Fret is a mechanism by which energy is transferred from one fluorophore to another, and widely used to estimate distances between probes on the scale of 2 to 10 nm.
The aims of this project were to engineer an intra molecular fret construct (cfp-yfp) with a linker containing a trypsin cleavage site was prepared to check the absorption and fluorescence spectra. Then the fret efficiency was determined by proteolysis assay and acceptor photo bleaching by a laser. On removal of the acceptor the peak of the donor fluorescence was increased in both cases. However, the efficiency of fret in the photo bleaching experiment (0.25) was much lower than the efficiency of fret obtained from the proteolysis assay (0.47). This suggests that the laser destroyed donor cfp to some extent, and therefore this method should not be used in cell biology experiments without appropriate correction.
Keywords: FRET, GFP, CFP, YFP, Photo bleaching, and Proteolysis assay.
Title: GFP FRET PROBE
Author: Iman Ajeyan AlAjeyan, Prof. Clive Bagshaw
International Journal of Healthcare Sciences
ISSN 2348-5728 (Online)
Research Publish Journals
