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Yeast Two-hybrid Assay for Detecting Protein Interaction   

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摘要:酵母双杂交系统可进行两个蛋白的互作分析。真核生物的位点特异性转录激活因子通常含有两个不同的结构域:DNA结合结构域 (DNA-binding domain) 和转录激活结构域 (transcription-activating domain)。前者可识别DNA上的特异序列,并使转录激活结构域定位于所调节基因的上游,转录激活结构域可同转录复合体的其他成分作用, 启动它所调节基因的转录。两个结构域可在适当部位打开且具有各自的功能,当两结构域通过适当的途径在空间上接近时其激活转录的能力可被恢复。将两待检测的蛋白分别与BD和AD融合并导入酵母菌株中,通过营养缺陷筛选可验证两个蛋白之间是否存在相互作用。

关键词: 蛋白互作, 酵母双杂交

材料与试剂

  1. 枪头
  2. 离心管
  3. ssDNA (10 mg/ml)
  4. 质粒DNA
  5. AH109菌液
  6. 酵母感受态
  7. DMSO
  8. PEG4000 (50%)
  9. 10x LiAc (1 M)
  10. Tris-HCl
  11. EDTA (pH7.5)
  12. YPD
  13. 0.2% Ade
  14. 琼脂粉
  15. SD-base
  16. SD/-leu/-trp
  17. SD/-ade/-leu/-trp/-his
  18. 10x TE (见溶液配方)
  19. 1x TE/LiAc (见溶液配方)
  20. PEG/LiAc (见溶液配方)
  21. 液体和固体YPDA培养基 (1 L) (见溶液配方)
  22. 营养缺陷型培养板 (1 L) (见溶液配方)
    1)
    二缺培养板
    2)
    四缺培养板

仪器设备

  1. 三角瓶
  2. 移液枪
  3. 涡旋振荡器
  4. 水浴锅
  5. 分光光度计
  6. 离心机
  7. 摇床
  8. 培养箱
  9. 高压灭菌锅

实验步骤

  1. 取少量AH109保存菌液,划线于固体YPDA培养板上;
  2. 28 °C培养2-4天,待长出直径2-3 mm的酵母单菌落;
  3. 挑取大而圆的酵母单菌落于干净离心管中,加1 ml 液体YPDA培养基涡旋混匀;
  4. 将上述菌液转入装有10 ml 液体YPDA培养基的三角瓶中,28 °C,250 rpm摇菌至OD600 = 1.4-1.8;
  5. 吸取1 ml菌液至装有100 ml 液体YPDA培养基的三角瓶中,摇至OD600 = 0.6-0.8;
  6. 将100 ml菌液分为两管,每管50 ml,室温1,000 x g离心5 min收集菌体,用25 ml的灭菌超纯水轻轻重悬;
  7. 室温1,000 x g离心5 min收集菌体,弃上清,重悬菌液于1.5 ml 1x TE/LiAc;
  8. 向干净离心管中依次加入下列成分:ssDNA 10 μl,质粒DNA 0.1 μg,酵母感受态100 μl,PEG/LiAc 600 μl;
  9. 涡旋1 min,使转化体系完全混匀;
  10. 30 °C的水浴30 min,加入70 μl DMSO;
  11. 再放入42 °C的水浴热击15 min,冰上冷却2 min;
  12. 室温2,000 x g离心10 s,弃上清;
  13. 加入1 ml的无菌水轻轻重悬沉淀;
  14. 吸200 μl转化混合物涂布在营养缺陷型培养板上;
  15. 28 °C培养2-4天观察并鉴定结果。

溶液配方

  1. 10x TE
    Tris-HCl
    0.1 M
    EDTA (pH7.5)
    10 mM
  2. 1x TE/LiAc
    10x TE
    1 ml
    10x LiAc
    1 ml
    ddH2O
    8 ml
  3. PEG/LiAc
    10x TE
    1 ml
    10x LiAc
    1 ml
    PEG4000 (50%)
    8 ml
  4. 液体和固体YPDA培养基 (1 L)
    50 g YPD溶于1 L去离子水加入15 ml 0.2% Ade,放入高压灭菌中进行灭菌;若配制培养板,再加入琼脂粉15 g
  5. 营养缺陷型培养板 (1 L)
    1)
    二缺培养板
    SD-base
    26.7 g
    SD/-leu/-trp0.64 g
    琼脂粉
    15 g
    2)
    四缺培养板
    SD-base
    26.7 g
    SD/-ade/-leu/-trp/-his0.60 g
    琼脂粉
    15 g
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Copyright: © 2018 The Authors; exclusive licensee Bio-protocol LLC.
引用格式:毛文文 , 魏灵芝 , 李冰冰 , 贾文锁 . (2018). 酵母双杂交技术检测蛋白互作. Bio-101: e1010223. DOI: 10.21769/BioProtoc.1010223.
How to cite: Mao, W. W., Wei, L. Z., Li, B. B. and Jia, W. S. (2018). Yeast Two-hybrid Assay for Detecting Protein Interaction. Bio-101: e1010223. DOI: 10.21769/BioProtoc.1010223.
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