多聚甲醛固定对荧光蛋白分子间FRET效率的影响

Effect of Paraformaldehyde Fixation on the FRET Efficiency Calculation between Fluorescent Proteins

目的：研究多聚甲醛固定对利用荧光共振能量转移（fluorescence resonance energy transfer，FRET）检测细胞中蛋白质相互作用的影响，解决运动能力较强的细胞中FRET效率检测的问题。方法：选用两个已知能够相互作用的蛋白分子TRA和TRB，将荧光蛋白ECFP和EYFP的编码基因通过融合PCR分别标记在其C端；将两个融合基因共转染靶细胞，一组细胞经低浓度（0．5％）多聚甲醛短时（0．5～1h）固定，另一组不固定，利用激光共聚焦扫描显微镜检测两个融合蛋白之间的FRET效率，比较其在两组细胞之间的差异情况。结果：经过统计学分析，在活细胞和经低浓度多聚甲醛短时间固定的细胞中，ECFP与EYFP之间的FRET效率没有显著差异。结论：低浓度短时间的多聚甲醛固定对于荧光蛋白分子之间的相互作用没有显著的影响，因此对于运动能力过强的细胞可以固定后再进行FRET检测。

Objective ： To investigate the effect of paraformaldehyde fixation on measuring the protein-protein interaction by fluorescence resonance energy transfer （FRET） to resolve the problem of FRET efficiency calculation in excess-movement cells. Methods： The C terminals of TCR α chain （TRA） and TCR β chain （TRB） genes, which were ideal for protein-protein interaction research, were fused with ECFP and EYFP gene respectively by fusion PCR and transferred into target cell. A grou Pcells were fixed in paraformaldehyde （0.5%） for 0.5 - 1 h and another left alive, then these cells were subject to ECFP/EYFP FRET calculation with confocal laser scanning microscope. The ECFP/EYFP FRET efficiencies in live and fixed cell were analyzed and compared. Results： There is no significant statistical difference between the ECFP/EYFP FRET efficiencies of live cell and cell fixed with lower paraformaldehyde concentration and shorter incubation time. Conclusion： fixation with low-concentration paraformaldehyde and short-time incubation has no distinct influence on measuring protein-protein interaction, and facilitated the FRET ealeulation in excess-movement cells.