MCF-7 细胞中的雌激素受体（ER）染色质免疫共沉淀

Pia Giovannelli; Gabriella Castoria; Antimo Migliaccio

doi:10.21769/BioProtoc.831

Improve Research Reproducibility A Bio-protocol resource

提交稿件
订阅
CN
- EN - English
- CN - 中文

Peer-reviewed

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 email

Gabriella Castoria

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: 10551

评审: Lin Fang

下载 PDF

Q&A

引用

Cited by

参见作者原研究论文

The authors used this protocol in:

Cover of Oncogene, featuring study using the protocol.

Nov 2012

实验方案合集

Cell Imaging - A Special Collection for Cell Bio 2023

相关实验方案

利用叠氮丙氨酸标记和流式细胞分析测定细胞周期中蛋白的合成

Koshi Imami and Tomoharu Yasuda

2019年04月20日 8779 阅读

Vangl 蛋白的泛素化和磷酸化分析

Di Feng [...] Bo Gao

2022年10月20日 2564 阅读

通过CRISPR-Cas9介导的HiBiT标签对高度同源蛋白水平进行异构体特异性半定量测定

Kristina Seiler [...] Mario P. Tschan

2023年07月20日 1509 阅读

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)

Breast cancer (乳腺癌)

ChIP (炸薯条)

MCF-7 cells (MCF-7细胞)

Transcription factor (转录因子)

Materials and Reagents

Fetal Bovine Serum (FBS) (Life Technologies, Gibco^®, catalog number: 10270 )
200 mM L-Glutamine (100x) (Life Technologies, Gibco^®, catalog number: 25030 )
Penicillin-Streptomycin (100x) (Life Technologies, Gibco^®, catalog number: 15140-122 )
Hydrocortisone (Sigma-Aldrich, catalog number: H-0888 )
Insulin (Human, recombinant) (F. Hoffmann-La Roche, catalog number: 11376497001 )
Estradiol (Beta-estradiol) (Sigma-Aldrich, catalog number: E8875 )
Protease inhibitors cocktail tablets (LAP tablets) (F. Hoffmann-La Roche, catalog number: 11836153001 )
Protein A-agarose fast flow (Sigma-Aldrich, catalog number: P3476 )
Formaldeyde (Sigma-Aldrich)
Salmon sperm DNA
DPBS (Sigma-Aldrich, catalog number: D5652 )
Proteinase K (F. Hoffmann-La Roche, catalog number: 03115879001 )
Qiagen PCR purification kit
Immunoprecipitating antibody (ER-alpha) (Sigma-Aldrich, catalog number: E1396 )
Primers CCDN1 locus (-1039 to 770 bp): Fw(-1039) (AACAAAACCAATTAGGAACCTT), Rv(-770) (ATTTCCTTCATCTTGTCCTTCT)
Primers CCDN1 promoter (-235 to -53 bp): Fw(-235) (TATGAAAACCGGACTACAGG), and Rv(-53) (CTGTTGTTAAGCAAAGATCAAAG)
Charcoal dextran stripped serum (CSS) (see Recipes)
Phenol Red-free Dulbecco’s Modified Medium (DMEM with PR) (Life Technologies, Gibco^®, catalog number: 31885 ) (for MCF-7 cells) (see Recipes)
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)
50x LAP (Protease inhibitor cocktail) (see Recipes)
DTT solution (see Recipes)
Buffer I (see Recipes)
Buffer II (see Recipes)
Buffer III or cell lysis buffer (see Recipes)
Buffer IV (see Recipes)
TSE I buffer (see Recipes)
TSE II buffer (see Recipes)
TSE III buffer (see Recipes)
TE buffer (see Recipes)
Elution buffer (see Recipes)
PCR buffer (see Recipes)

Equipment

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

Procedure

English

中文翻译

文章信息

版权信息

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.