Fluorescence resonance energy transfer (FRET) http://www.microimage.com.cn (2008-03-07 14:59:52) a live cell research problems encountered

Fluorescence resonance energy transfer (FRET) http://www.microimage.com.cn/ (2008-03-07 14:59:52) a live cell research problems encountered:
Mutual combination of proteins or other molecules in living cells and the location is the key issue to understand their function. To answer this question, the protein marked with different fluorophores. However, the resolution of optical microscopy to protein detection accuracy is limited to around about 0.2μm. To study the physical interactions of the protein components of high resolution.
Second, what is of FRET?
FRET is the use of non-radiation methods, the close proximity (1-10 nm) in the donor and recipient each other, the transfer of the photon from an excited fluorophore (donor) to another fluorophore (acceptor) . Using FRET, can be solved more than the resolution limit of optical microscopy of molecular relative proximity to display (for example) (1) the molecular interaction between the two protein components; (2) within a molecule (such as enzyme activity, DNA / RNA changes in the structure of the form); (3) ion concentration such as the CFP-of YFP Cameleon Special FRET-tools

                
                          CFP by light excitation after emission CFP by light excitation, but did not launch Xiao Guang.
                         CFP from YFP distance greater than 10nm the CFP and YFP is very close (1-10 nm)
                         YFP excitation has not been, so do not emit light CFP did not excited by light, but the light emitted

Third, FRET principle
By the excited fluorophore (the donor) will be excited by the static can be transferred to a light absorbing molecule (receptor). The transfer of non-radioactive, mainly because between the donor and acceptor dipole - dipole interaction. Dipole reaction in the excited state donor to the non-excitation energy transfer to the next receptor. This theory is based on the excited fluorescent molecules as oscillating dipole, and the same oscillation frequency of the other dipole energy exchange.
Only a few fluorophores suitable for FRET experiments, because, in addition to other necessary conditions (such as dipole orientation, adequate fluorescence lifetime), the donor emission spectrum must be the receptor excitation spectra overlap. Known FRET CFP / of YFP, BFP / GFP, GFP / rhodamine, FITC/Cy3


CFP / of YFP of FRET energy diagram:
CFP (donor) was excited, but most of the energy does not cause green emitters; but transferred to the of YFP (acceptor); therefore, the resulting emission is mainly yellow.

Fourth, the FRET applications
Such as green fluorescent protein (GFP) fluorescent proteins (FPs) are very attractive for FRET experiments. They can be genetically fused to the relevant proteins and expressed in cells, making them excellent reporter system for gene expression and protein localization in living cells. Can provide enhanced FP variants with different spectral characteristics.
Cyan-colored CFP as donor, the yellow YFP as acceptor is the most suitable for living cells for FRET experiments, the emission spectrum of CFP partially overlaps the YFP excitation spectrum.

 
When they are close enough to stimulate the absorption wavelength of CFP, the CFP chromophore will be high energy efficient resonant transfer to YFP the chromophore, the emission fluorescence of CFP diminished or disappeared, the main launch will be YFP fluorescence. The energy conversion efficiency between the two chromophore and the distance of space between them is inversely proportional to the power of 6, the change of spatial location is very sensitive [1-2]. For example, to study two proteins, the interaction between a and b, according to the FRET principle to build a fusion protein, this fusion protein composed of three parts: the CFP (cyan fluorescent protein), protein b, of YFP (yellow fluorescent protein). CFP absorption wavelength of 433nm as the excitation wavelength, smart experimental design, so that when the protein a and b does not interact very far away from the CFP and YFP can not occur fluorescence resonance energy transfer, and thus detected by the CFP emission wavelength of 476nm fluorescence; But when the protein a and b interact, protein b by a role in protein conformational changes, the CFP and YFP fully close to the occurrence of fluorescence resonance energy transfer detected YFP of the emission wavelength of 527nm fluorescence (Figure 1). The gene encoding this fusion protein by transgenic technology expressed in the cell, so that under physiological conditions of living cells to study protein - protein interactions