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水稻叶片细胞膜蛋白分离   

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实验原理:细胞膜蛋白由于是嵌在膜中,水溶性较差。基本方法是用不同的离心速度去掉胞质蛋白等,用去污剂把细胞膜蛋白从膜中释放出来。
实验目的:分离水稻叶片细胞膜蛋白,可用于Western blot检测某个蛋白质是否是细胞膜蛋白。由于分离细胞膜蛋白一直比较困难,目前有一些试剂盒专门用于细胞膜蛋白的分离。此方法分离到的水稻细胞膜蛋白质是含有所有膜组分的总细胞膜蛋白质,不适用于水稻质膜蛋白或其它亚细胞器膜蛋白的分离。

关键词: 水稻叶片, 超速离心机, 总膜蛋白

材料与试剂

  1. 水稻叶片
  2. 液氮
  3. Tris-HCl (Trizma hydrochloride) (Sigma, catalog number: T5941)
  4. NaCl (Sodium chloride) (Sigma, catalog number: S3014)
  5. Sucrose (Sigma, catalog number: S9378)
  6. PMSF (PHenylmethanesulfonyl fluoride) (Sigma, catalog number: P7626)
  7. EDTA (Ethlenediaminetetraacetic acid) (Sigma, catalog number: E6758)
  8. Triton X-100 (Sigma, catalog number: T8787)
  9. Protease inhibitor cocktail (Sigma, catalog number: P9599)
  10. 蛋白抽提液 (见溶液配方)
  11. 重悬缓冲液 (见溶液配方)

仪器设备

  1. 研钵
  2. 天平
  3. Type 70 Ti Rotor离心管
  4. Beckman超速离心机 (LE-80K)

实验步骤

  1. 取水稻叶片5 g,经液氮研磨后,加入3倍体积蛋白抽提液 (溶液配方1),在4 °C混匀30 min。
  2. 于4 °C,15,000 x g离心30 min,吸取上清于一个新的离心管中。
  3. 重复步骤2一次。
  4. 将上清转移至Beckman超速离心机专用Type 70 Ti Rotor离心管中。于4 °C,100,000 x g离心2 h。上清为可溶蛋白,下部沉淀为水稻总膜蛋白。将上清弃之,然后用重悬缓冲液 (溶液配方2)于4 °C溶解下部沉淀2 h左右,即为水稻叶片总膜蛋白。
  5. 按照Bradford方法测定蛋白质浓度 (Bradford, 1976)。于-70 °C保存待用。

溶液配方

  1. 蛋白抽提液
    50 mM Tris-HCl, pH 7.5
    150 mM NaCl
    50 mM Sucrose
    1 mM PMSF
    1 mM EDTA
    1 % Triton X-100
    1x protease inhibitor cocktail
  2. 重悬缓冲液
    50 mM Tris-HCl, pH 7.5
    150 mM NaCl
    50 mM Sucrose
    1 mM EDTA
    1% Triton X-100
    1x protease inhibitor cocktail

参考文献

  1. Bradford, M. M. (1976). A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. Anal Biochem 72: 248-254.
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Copyright: © 2018 The Authors; exclusive licensee Bio-protocol LLC.
引用格式:袁猛. (2018). 水稻叶片细胞膜蛋白分离. Bio-101: e1010124. DOI: 10.21769/BioProtoc.1010124.
How to cite: Yuan, M. (2018). Isolation of Cell Membrane Proteins from Rice Leaves. Bio-101: e1010124. DOI: 10.21769/BioProtoc.1010124.
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