免疫共沉淀(Co-IP)检测草莓果实中蛋白互作   

How to cite Favorites Q&A Share your feedback Cited by

摘要:免疫共沉淀是以抗体和抗原之间的专一性作用为基础的用于研究蛋白质相互作用的方法。当细胞在非变性条件下被裂解时,完整细胞内存在的许多蛋白质与蛋白质间的相互作用也被保留下来。用预先固化在beads上的蛋白质A的抗体免疫沉淀A蛋白,那么与A蛋白在体内结合的蛋白质B也能一起沉淀下来,再通过蛋白变性分离,对B蛋白进行检测,进而证明两者间的相互作用。

关键词: 草莓果实, Co-IP, 蛋白互作

材料与试剂

  1. 枪头
  2. 离心管
  3. 草莓大绿果或白果
  4. 菌株 (pSPYNE和pSPYCE)
  5. 液氮
  6. Agarose protein A+G beads
  7. SDS Loading Buffer
  8. 磷酸缓冲液 (pH 7.8)
  9. EDTA
  10. 甘油
  11. TritonX-100
  12. DTT
  13. 蛋白裂解buffer (见溶液配方)

仪器设备

  1. 移液枪
  2. 注射器
  3. 研磨用具
  4. 涡旋振荡器
  5. 离心机

实验步骤

  1. 将待测蛋白菌液按1:1混合注射于草莓大绿果或白果 (图1) 中,此时注射菌液蛋白表达效果较好,3天后取样 (所用菌株为pSPYNE, pSPYCE);


    图1. 草莓果实大绿果和白果时期

  2. 提取草莓果实蛋白,液氮中磨样充分,加入裂解buffer,涡旋混匀,冰上放置30 min使其充分裂解,4 °C,13,800 x g离心10 min;
  3. 取上清于2 ml离心管中,另取50 μl为input;
  4. 离心管中按比例加入抗体,室温孵育1 h或4 °C过夜;
  5. 按1:10比例加入agarose protein A + G,室温3 h,4 °C,1,000 x g离心收集beads,并用预冷的裂解buffer清洗beads约5次;
  6. 洗完最后一次,去掉上清后,向beads中加入SDS Loading Buffer至终浓度为1x,混匀后加热煮沸5 min,冷却后,13,800 x g离心取适量点样;
  7. Western blot检测蛋白,具体步骤参见毛文文等,(2018)。

溶液配方

  1. 蛋白裂解buffer
    磷酸缓冲液 (pH 7.8)
    1x
    EDTA
    1 mM
    甘油 10%
    TritonX-100
    0.5%
    DTT 1 mM

参考文献

  1. 毛文文,魏灵芝,李冰冰,贾文锁. (2018). 草莓果实蛋白提取和Western Blot. Bio-101 e1010222. Doi: 10.21769/BioProtoc.1010222.
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). 免疫共沉淀(Co-IP)检测草莓果实中蛋白互作. Bio-101: e1010224. DOI: 10.21769/BioProtoc.1010224.
How to cite: Mao, W. W., Wei, L. Z., Li, B. B. and Jia, W. S. (2018). Protein-protein Interaction Detection in Strawberry Fruit by Co-IP Assay. Bio-101: e1010224. DOI: 10.21769/BioProtoc.1010224.
Q&A

Please login to post your questions/comments. Your questions will be directed to the authors of the protocol. The authors will be requested to answer your questions at their earliest convenience. Once your questions are answered, you will be informed using the email address that you register with bio-protocol.
You are highly recommended to post your data including images for the troubleshooting.

You are highly recommended to post your data (images or even videos) for the troubleshooting. For uploading videos, you may need a Google account because Bio-protocol uses YouTube to host videos.