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Estradiol Receptor (ER) Chromatin Immunoprecipitation in MCF-7 Cells

MCF-7 细胞中的雌激素受体（ER）染色质免疫共沉淀

Pia Giovannelli Pia Giovannelli
Gabriella Castoria Gabriella Castoria
Antimo Migliaccio Antimo Migliaccio

发布: 2013年07月20日第3卷第14期 DOI: 10.21769/BioProtoc.831 浏览次数: 11220

评审: Lin Fang

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Abstract

Steroid hormone receptors, for example estradiol receptor, act like transcription factors. In the cell, steroids bind to a specific receptor. Upon ligand binding, many steroid receptors dimerize and enter nuclei where they bind specific DNA sequences called Hormone Responsive Elements (HRE) and regulate gene transcription. ER is able to bind DNA sites that are not Estrogen Responsive Elements (ERE) so regulating also the transcription of genes that are not classically controlled by estrogens.

Keywords: Estradiol Receptor (雌二醇受体) search Breast cancer (乳腺癌) search ChIP (炸薯条) search MCF-7 cells (MCF-7细胞) search Transcription factor (转录因子) search

Materials and Reagents

  1. Fetal Bovine Serum (FBS) (Life Technologies, Gibco®, catalog number: 10270 )
  2. 200 mM L-Glutamine (100x) (Life Technologies, Gibco®, catalog number: 25030 )
  3. Penicillin-Streptomycin (100x) (Life Technologies, Gibco®, catalog number: 15140-122 )
  4. Hydrocortisone (Sigma-Aldrich, catalog number: H-0888 )
  5. Insulin (Human, recombinant) (F. Hoffmann-La Roche, catalog number: 11376497001 )
  6. Estradiol (Beta-estradiol) (Sigma-Aldrich, catalog number: E8875 )
  7. Protease inhibitors cocktail tablets (LAP tablets) (F. Hoffmann-La Roche, catalog number: 11836153001 )
  8. Protein A-agarose fast flow (Sigma-Aldrich, catalog number: P3476 )
  9. Formaldeyde (Sigma-Aldrich)
  10. Salmon sperm DNA
  11. DPBS (Sigma-Aldrich, catalog number: D5652 )
  12. Proteinase K (F. Hoffmann-La Roche, catalog number: 03115879001 )
  13. Qiagen PCR purification kit
  14. Immunoprecipitating antibody (ER-alpha) (Sigma-Aldrich, catalog number: E1396 )
  15. Primers CCDN1 locus (-1039 to 770 bp): Fw(-1039) (AACAAAACCAATTAGGAACCTT), Rv(-770) (ATTTCCTTCATCTTGTCCTTCT)
  16. Primers CCDN1 promoter (-235 to -53 bp): Fw(-235) (TATGAAAACCGGACTACAGG), and Rv(-53) (CTGTTGTTAAGCAAAGATCAAAG)
  17. Charcoal dextran stripped serum (CSS) (see Recipes)
  18. Phenol Red-free Dulbecco’s Modified Medium (DMEM with PR) (Life Technologies, Gibco®, catalog number: 31885 ) (for MCF-7 cells) (see Recipes)
  19. Phenol Red-free Dulbecco’s Modified Medium (DMEM w/o PR) (Life Technologies, Gibco®, catalog number: 11880 ) supplemented with Charcoal (for MCF-7 cells) (see Recipes)
  20. 50x LAP (Protease inhibitor cocktail) (see Recipes)
  21. DTT solution (see Recipes)
  22. Buffer I (see Recipes)
  23. Buffer II (see Recipes)
  24. Buffer III or cell lysis buffer (see Recipes)
  25. Buffer IV (see Recipes)
  26. TSE I buffer (see Recipes)
  27. TSE II buffer (see Recipes)
  28. TSE III buffer (see Recipes)
  29. TE buffer (see Recipes)
  30. Elution buffer (see Recipes)
  31. PCR buffer (see Recipes)

Equipment

  1. Sonicator (equipped with a 3 mm diameter tip) (Sonics & Materials Inc., model: Vibracell VC 130PB )
  2. 100 mm dishes
  3. Centrifuge (Eppendorf centrifuge, model: 5417R )
  4. Shaker
  5. PCR thermal cycler

Procedure

English
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文章信息

版权信息

© 2013 The Authors; exclusive licensee Bio-protocol LLC.

如何引用

Giovannelli, P., Castoria, G. and Migliaccio, A. (2013). Estradiol Receptor (ER) Chromatin Immunoprecipitation in MCF-7 Cells. Bio-protocol 3(14): e831. DOI: 10.21769/BioProtoc.831.

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分类

癌症生物学 > 通用技术 > 生物化学试验 > 蛋白质分析

生物化学 > 蛋白质 > 免疫检测 > 染色质免疫共沉淀（ChIP）

分子生物学 > DNA > DNA-蛋白质相互作用

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