甲醛固定对GFP发光特性的影响
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摘要
绿色荧光蛋白(GFP)能够作为报告分子对活体细胞中特定基因的时空表达进行实时追踪,因此广泛应用于生物学研究领域。在用GFP对细胞活动进行追踪的实验中,常有无法在取样后及时对样品进行荧光观察的情况,此时需要先将样品进行固定以便对其进行观测。然而,不恰当的细胞固定方法会导致胞内GFP荧光信号强度减弱、位置改变等后果。甲醛是最常用的细胞固定剂,也常被用于固定表达GFP蛋白的细胞样品。但对甲醛固定GFP样品的报道多是针对于真核细胞,且固定效果也存在较大差异。文章系统地探索了甲醛浓度、固定时间、固定缓冲液种类对两种细菌(E.coli及鱼腥蓝细菌Anabaena PCC7120)胞内GFP信号的影响。结果显示,较低浓度(≤1%)的甲醛处理2h后,细胞的荧光强度在1d后仍可保持80%以上,胞内荧光点无弥散现象发生。具有相近pH的几种常见缓冲液对荧光强度的影响无显著差异。随着甲醛浓度的增加、固定时间的延长、溶液pH的增加(中性至偏碱性),细胞中的荧光强度会逐渐降低。
Green fluorescent protein(GFP)is widely used in biological researches for real-time detection of gene expression and subcellular localization in living cells.GFP samples need to be fixed for later observation in case they are not able to be observed in time.However,improper fixation methods often lead to loss and/or mislocation of fluorescence signal.Formaldehyde is the most widely chosen reagent for fixation of many types of cells and tissues.It has also been used to fix GFP-expressing samples(mostly eukaryotic cells),but the performance varied much as reported in different literatures.This study explores the effects of formaldehyde concentration,fixation time and buffer on cellular GFP fluorescence for E.coli or AnabaenaPCC7120.Our results showed that after the treatment with lower concentrations of formaldehyde(≤1%)for 2hours,the cellular GFP fluorescence could retain more than 80% of the original intensity and its subcellular location was well preserved within one day.Several common buffers resulted similar fixation performance when they were used at close pH.However,the intensity of intracellular GFP fluorescence gradually decreased along with the increases of formaldehyde concentration,fixation time and the pH of solution.
Green fluorescent protein(GFP)is widely used in biological researches for real-time detection of gene expression and subcellular localization in living cells.GFP samples need to be fixed for later observation in case they are not able to be observed in time.However,improper fixation methods often lead to loss and/or mislocation of fluorescence signal.Formaldehyde is the most widely chosen reagent for fixation of many types of cells and tissues.It has also been used to fix GFP-expressing samples(mostly eukaryotic cells),but the performance varied much as reported in different literatures.This study explores the effects of formaldehyde concentration,fixation time and buffer on cellular GFP fluorescence for E.coli or AnabaenaPCC7120.Our results showed that after the treatment with lower concentrations of formaldehyde(≤1%)for 2hours,the cellular GFP fluorescence could retain more than 80% of the original intensity and its subcellular location was well preserved within one day.Several common buffers resulted similar fixation performance when they were used at close pH.However,the intensity of intracellular GFP fluorescence gradually decreased along with the increases of formaldehyde concentration,fixation time and the pH of solution.
引文
[1]张广峰,陈祥贵,帅培强,等.细胞固定剂对GFP发光特性影响的研究[J].化学与生物工程,2010,27(12):38-40.
[2]Zhang C,Xing X H.Fluorescent proteins as a visible molecular signal for rapid quantification of bioprocesses:potential and challenges[J].Chinese Journal of Chemical Engineering,2010,18(5):863-869.
[3]薛启汉.绿色荧光蛋白(GFP)的特性及其在分子生物学研究中的应用[J].江苏农业学报,1999,15(1):52-58.
[4]Ulrike S,Freark D,Klaas A S,et al.Immunolabeling artifacts and the need for live-cell imaging[J].Nature Methods,2012,9(2):152-158.
[5]Joosen L,Hink M A,Gadella T W,et al.Effect of fixation procedures on the fluorescence lifetimes of Aequorea victoria derived fluorescent proteins[J].Journal of Microscopy,2014,256(3):166-176.
[6]Jan W B,Mark A H,Arievan H,et al.effects of refractive index and viscosity on fluorescence and anisotropy decays of enhanced cyan and yellow fluorescent proteins[J].Journal of Fluorescence,2005,15(2):153-160.
[7]李荣芳,袁慧,刘定干.甲醇固定导致绿色荧光蛋白的荧光消失[J].细胞生物学杂志,2007,29:303-304.
[8]Roland B,Irene H L,Thomas M J.Comparison of fixation protocols for adherent cultured cells applied to agfp fusion protein of the epidermal growth factor receptor[J].Cytometry,1999,35(4):353-362.
[9]Straight A F.Fluorescent protein applications in microscopy[J].Methods in Cell Biology,2007,114:93-113.
[10]伍艳辉,白素松,谭露璐,等.多聚甲醛的解聚实验研究[J].实验室研究与探索,2008,27(6):69-71.
[11]武晓琼,闫荟,杨锋,等.正交试验设计优选多聚甲醛的解聚方法[J].中国药业,2014,23(14):62-64.
[12]刘双又,李小兰,肖薇,等.绿色荧光蛋白细胞的保存及细胞周期分析[J].华中科技大学学报(医学版),2007,36(6):798-800,820.
[13]鞠传丽,王东,孔冬冬,等.绿色荧光蛋白在植物细胞生物学中的应用[J].生命的化学,2001,21(6):445-447.
[14]储军,施华,杨杰,等.分子与细胞事件的光学可视化——解读2008年诺贝尔化学奖[J].生物化学与生物物理进展,2008,35(10):1104-1111.
[2]Zhang C,Xing X H.Fluorescent proteins as a visible molecular signal for rapid quantification of bioprocesses:potential and challenges[J].Chinese Journal of Chemical Engineering,2010,18(5):863-869.
[3]薛启汉.绿色荧光蛋白(GFP)的特性及其在分子生物学研究中的应用[J].江苏农业学报,1999,15(1):52-58.
[4]Ulrike S,Freark D,Klaas A S,et al.Immunolabeling artifacts and the need for live-cell imaging[J].Nature Methods,2012,9(2):152-158.
[5]Joosen L,Hink M A,Gadella T W,et al.Effect of fixation procedures on the fluorescence lifetimes of Aequorea victoria derived fluorescent proteins[J].Journal of Microscopy,2014,256(3):166-176.
[6]Jan W B,Mark A H,Arievan H,et al.effects of refractive index and viscosity on fluorescence and anisotropy decays of enhanced cyan and yellow fluorescent proteins[J].Journal of Fluorescence,2005,15(2):153-160.
[7]李荣芳,袁慧,刘定干.甲醇固定导致绿色荧光蛋白的荧光消失[J].细胞生物学杂志,2007,29:303-304.
[8]Roland B,Irene H L,Thomas M J.Comparison of fixation protocols for adherent cultured cells applied to agfp fusion protein of the epidermal growth factor receptor[J].Cytometry,1999,35(4):353-362.
[9]Straight A F.Fluorescent protein applications in microscopy[J].Methods in Cell Biology,2007,114:93-113.
[10]伍艳辉,白素松,谭露璐,等.多聚甲醛的解聚实验研究[J].实验室研究与探索,2008,27(6):69-71.
[11]武晓琼,闫荟,杨锋,等.正交试验设计优选多聚甲醛的解聚方法[J].中国药业,2014,23(14):62-64.
[12]刘双又,李小兰,肖薇,等.绿色荧光蛋白细胞的保存及细胞周期分析[J].华中科技大学学报(医学版),2007,36(6):798-800,820.
[13]鞠传丽,王东,孔冬冬,等.绿色荧光蛋白在植物细胞生物学中的应用[J].生命的化学,2001,21(6):445-447.
[14]储军,施华,杨杰,等.分子与细胞事件的光学可视化——解读2008年诺贝尔化学奖[J].生物化学与生物物理进展,2008,35(10):1104-1111.