果蝇脂类薄层层析   

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摘要:本实验通过薄层层析的方法分离果蝇组织中的脂类。

关键词: 果蝇, 薄层层析, 脂类

材料与试剂

  1. 滤纸
  2. 铅笔
  3. 毛细管
  4. 展开剂:不同的展开剂对脂的分辨率和条带不同,应参考自己的样品和分析需求测试合适的展开剂。
    1)
    Chloroform (氯仿)/methanol (甲醇)/acetic acid (醋酸)/formic acid (甲酸)/water (水) (70:30:12:4:2) (Arikketh等,2008)
    2)
    Chloroform (氯仿)/ethanol (乙醇)/water (水)/trimethylamine (三甲氨) (30:35:7:35)
    3)
    Chloroform (氯仿)/methanol (甲醇)/acetic acid (醋酸)/water (水) (50:28:4:8) (Nigou和Besra,2002;Vaden等,2005;Tamura等,2013)
    4)
    Chloroform (氯仿)/methanol (甲醇)/25% ammonia (氨水) (65:35:5) (Aaltonen等,2016)
    5)
    Chloroform (氯仿)/methylacetate (醋酸甲酯)/1-propanol (1-丙醇)/methanol (甲醇)/0.25% KCl (氯化钾) (25:25:25:10:9) (Miyata等,2016)
    6)
    Chloroform (氯仿)/hexane (己烷)/methanol (甲醇)/acetic acid (醋酸) (50:30:10:5) (Tasseva等,2013)
  5. 碘晶体 (化学纯即可)

仪器设备

  1. 烘箱
  2. 层析缸
  3. 层析板 (TLC silica gel 60 F254 plate) (Merck, catalog number: 1.05729.0001)
  4. 通风橱
  5. 含有碘晶体的容器

实验步骤

  1. 装配层析缸,放入1到2片滤纸,将其紧贴缸壁 (图1)。
  2. 倒入80~100 ml展开剂 (材料与试剂4) [可以用1) ~ 6)不同展开剂],盖紧层析缸盖,平衡缸内空气30~60分钟。


    图1. 层析缸装配实物图

  3. 使用铅笔在层析板底部2 cm处绘制水平直线,并设计样品点样位置。在距离层析板顶部2 cm处绘制水平直线,作为层析流动相终点处的位置。
  4. 将层析板放置在70 °C烘箱中干燥至少15分钟。
  5. 用毛细管吸取含有待分析的脂样品的有机溶液20 μl,在点样位置上样,全完上样后将层析板置于室温或用吹风机干燥。
    注:组织脂样品按照参考文献提供的方法提取,最终由氯仿/甲醇 (1:2) 重新溶解。
  6. 将干燥后的层析板放置到层析缸中。注意不要让展开剂液面超过用铅笔绘制的水平线。
  7. 当展开剂流动相达到层析板上部绘制的水平线后取出,然后在通风橱内将层析板干燥。
  8. 将层析板放置到含有碘晶体的容器中染色 (图2),并拍照扫描。


    图2. 层析板置放于染色容器示意图

参考文献

  1. Aaltonen, M. J., Friedman, J. R., Osman, C., Salin, B., di Rago, J. P., Nunnari, J., Langer, T. and Tatsuta, T. (2016). MICOS and phospholipid transfer by Ups2-Mdm35 organize membrane lipid synthesis in mitochondria. J Cell Biol 213(5): 525-534.
  2. Arikketh, D., Nelson, R. and Vance, J. E. (2008). Defining the importance of phosphatidylserine synthase-1 (PSS1): unexpected viability of PSS1-deficient mice. J Biol Chem 283(19): 12888-12897.
  3. Miyata, N., Watanabe, Y., Tamura, Y., Endo, T. and Kuge, O. (2016). Phosphatidylserine transport by Ups2-Mdm35 in respiration-active mitochondria. J Cell Biol 214(1): 77-88.
  4. Nigou, J. and Besra, G. S. (2002). Cytidine diphosphate-diacylglycerol synthesis in Mycobacterium smegmatis. Biochem J 367(Pt 1): 157-162.
  5. Tamura, Y., Harada, Y., Nishikawa, S., Yamano, K., Kamiya, M., Shiota, T., Kuroda, T., Kuge, O., Sesaki, H., Imai, K., Tomii, K. and Endo, T. (2013). Tam41 is a CDP-diacylglycerol synthase required for cardiolipin biosynthesis in mitochondria. Cell Metab 17(5): 709-718.
  6. Tasseva, G., Bai, H. D., Davidescu, M., Haromy, A., Michelakis, E. and Vance, J. E. (2013). Phosphatidylethanolamine deficiency in mammalian mitochondria impairs oxidative phosphorylation and alters mitochondrial morphology. J Biol Chem 288(6): 4158-4173.
  7. Vaden, D. L., Gohil, V. M., Gu, Z. and Greenberg, M. L. (2005). Separation of yeast phospholipids using one-dimensional thin-layer chromatography. Anal Biochem 338(1): 162-164.
Copyright: © 2019 The Authors; exclusive licensee Bio-protocol LLC.
引用格式:杨晓, 黄勋. (2019). 果蝇脂类薄层层析. Bio-101: e1010282. DOI: 10.21769/BioProtoc.1010282.
How to cite: Yang, X. and Huang, X. (2019). Thin Layer Chromatography Analysis of Drosophila Lipid. Bio-101: e1010282. DOI: 10.21769/BioProtoc.1010282.
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