Effect of Paraformaldehyde Fixation on the FRET Efficiency Calculation between Fluorescent Proteins
- VernacularTitle:多聚甲醛固定对荧光蛋白分子间FRET效率的影响
- Author:
Hong-Wei SHAO
;
Wen-Feng ZHANG
;
Qing-Lian HU
;
Han SHEN
;
Feng-Lin WU
;
Shu-Lin HUANG
;
- Publication Type:Journal Article
- Keywords:
FRET ECFP/EYFP Fixation Paraformaldehyde
- From:
China Biotechnology
2006;0(07):-
- CountryChina
- Language:Chinese
-
Abstract:
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 ? chain(TRA)and TCR ? 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 0.5~1h 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 calculation in excess-movement cells.
