Revision 13

#8644

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Orders:

877-616-CELL (2355)

[email protected]

Support:

877-678-TECH (8324)

3 Trask Lane | Danvers | Massachusetts | 01923 | USA

For Research Use Only. Not for Use in Diagnostic Procedures.

Applications:
W, W-S, IP, IHC-P, ChIP, ChIP-seq, C&R, C&T

Reactivity:
H

Sensitivity:
Endogenous

MW (kDa):
66

Source/Isotype:
Rabbit IgG

UniProt ID:
#P03372

Entrez-Gene Id:
2099

Product Usage Information

For optimal ChIP and ChIP-seq results, use 5 μl of antibody and 10 μg of chromatin (approximately 4 x 106 cells) per IP. This antibody has been validated using SimpleChIP® Enzymatic Chromatin IP Kits.

The CUT&RUN dilution was determined using CUT&RUN Assay Kit #86652.

The CUT&Tag dilution was determined using CUT&Tag Assay Kit #77552.

Application Dilution
Western Blotting 1:1000
Simple Western™ 1:10 - 1:50
Immunoprecipitation 1:50
Immunohistochemistry (Paraffin) 1:600 - 1:2400
Chromatin IP 1:100
Chromatin IP-seq 1:100
CUT&RUN 1:100
CUT&Tag 1:100

Storage

Supplied in 10 mM sodium HEPES (pH 7.5), 150 mM NaCl, 100 µg/ml BSA, 50% glycerol and less than 0.02% sodium azide. Store at –20°C. Do not aliquot the antibody.

For a carrier free (BSA and azide free) version of this product see product #36911.

Specificity/Sensitivity

Estrogen Receptor alpha (D8H8) Rabbit Monoclonal Antibody recognizes endogenous levels of total ERα protein.

Source / Purification

Monoclonal antibody is produced by immunizing animals with a synthetic peptide corresponding to residues surrounding Val560 of human ERα protein.

Background

Estrogen receptor α (ERα), a member of the steroid receptor superfamily, contains highly conserved DNA binding and ligand binding domains (1). Through its estrogen-independent and estrogen-dependent activation domains (AF-1 and AF-2, respectively), ERα regulates transcription by recruiting coactivator proteins and interacting with general transcriptional machinery (2). Phosphorylation at multiple sites provides an important mechanism to regulate ERα activity (3-5). Ser104, 106, 118, and 167 are located in the amino-terminal transcription activation function domain AF-1, and phosphorylation of these serine residues plays an important role in regulating ERα activity. Ser118 may be the substrate of the transcription regulatory kinase CDK7 (5). Ser167 may be phosphorylated by p90RSK and Akt (4,6). According to the research literature, phosphorylation at Ser167 may confer tamoxifen resistance in breast cancer patients (4).

Species Reactivity

Species reactivity is determined by testing in at least one approved application (e.g., western blot).

Western Blot Buffer

IMPORTANT: For western blots, incubate membrane with diluted primary antibody in 5% w/v BSA, 1X TBS, 0.1% Tween® 20 at 4°C with gentle shaking, overnight.

Applications Key

W: Western Blotting W-S: Simple Western™ IP: Immunoprecipitation IHC-P: Immunohistochemistry (Paraffin) ChIP: Chromatin IP ChIP-seq: Chromatin IP-seq C&R: CUT&RUN C&T: CUT&Tag

Cross-Reactivity Key

H: Human

Trademarks and Patents

Cell Signaling Technology is a trademark of Cell Signaling Technology, Inc.

All other trademarks are the property of their respective owners. Visit cellsignal.com/trademarks for more information.

使用に関する制限

使用に関する制限

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研究専用 (For Research Use Only) またはこれに類似する表示がされた製品は、 いかなる目的についても FDA または外国もしくは国内のその他の規制機関により承認、認可または許可を受けていません。 お客様は製品を診断もしくは治療目的で使用してはならず、また、製品に表示された内容に違反する方法で使用してはなりません。 CST が販売または使用許諾する製品は、エンドユーザーであるお客様に対し、使途を研究および開発のみに限定して提供されるものです。 診断、予防もしくは治療目的で製品を使用することまたは製品を再販売 (単独であるか他の製品等の一部であるかを問いません) もしくはその他の商業的利用の目的で購入することについては、CST から別途許諾を得る必要があります。 お客様は以下の事項を遵守しなければなりません。(a) CST の製品 (単独であるか他の資材と一緒であるかを問いません) を販売、使用許諾、貸与、寄付もしくはその他の態様で第三者に譲渡したり使用させたりしてはなりません。また、商用の製品を製造するために CST の製品を使用してはなりません。(b) 複製、改変、リバースエンジニアリング、逆コンパイル、 分解または他の方法により製品の構造または技術を解明しようとしてはなりません。また、 CST の製品またはサービスと競合する製品またはサービスを開発する目的で CST の製品を使用してはなりません。(c) CST の製品の商標、商号、ロゴ、特許または著作権に関する通知または表示を除去したり改変したりしてはなりません。(d) CST の製品をCST 製品販売条件(CST’s Product Terms of Sale) および該当する書面のみに従って使用しなければなりません。(e) CST の製品に関連してお客様が使用する第三者の製品またはサービスに関する使用許諾条件、 サービス提供条件またはこれに類する合意事項を遵守しなければなりません。

Orders: 877-616-CELL (2355) [email protected] Support: 877-678-TECH (8324) [email protected] Web: cellsignal.com
For Research Use Only. Not for Use in Diagnostic Procedures.

Revision 13

Cell Signaling Technologyロゴ
Western blot analysis of extracts from ER-positive cell lines (MCF7, T-47D, ZR-75-1) and ER-negative cell lines (SK-BR-3 and MCF 10A) using Estrogen Receptor α (D8H8) Rabbit mAb (upper) or β-Actin (D6A8) Rabbit mAb #8457 (lower).
Western Blotting Image 1: Estrogen Receptor alpha (D8H8) Rabbit Monoclonal Antibody
Simple Western™ analysis of lysates (1 mg/mL) from MCF7 cells using Estrogen Receptor α (D8H8) Rabbit mAb #8644. The virtual lane view (left) shows the target band (as indicated) at 1:10 and 1:50 dilutions of primary antibody. The corresponding electropherogram view (right) plots chemiluminescence by molecular weight along the capillary at 1:10 (blue line) and 1:50 (green line) dilutions of primary antibody. This experiment was performed under reducing conditions on the Jess™ ​​​​​​​ Simple Western instrument from ProteinSimple, a BioTechne brand, using the 12-230 kDa separation module.
Western Blotting Image 1: Estrogen Receptor alpha (D8H8) Rabbit Monoclonal Antibody
Immunohistochemical analysis of paraffin-embedded human ductal breast carcinoma using Estrogen Receptor alpha (D8H8) Rabbit Monoclonal Antibody (left) compared to concentration-matched Rabbit (DA1E) Monoclonal Antibody IgG Isotype Control #3900 (right).
Immunohistochemistry Image 1: Estrogen Receptor alpha (D8H8) Rabbit Monoclonal Antibody
Orders: 877-616-CELL (2355) [email protected] Support: 877-678-TECH (8324) [email protected] Web: cellsignal.com
For Research Use Only. Not for Use in Diagnostic Procedures.

Revision 13

Cell Signaling Technologyロゴ
Immunohistochemical analysis of paraffin-embedded normal human uterus (left) and breast carcinoma (right) using Estrogen Receptor alpha (D8H8) Rabbit Monoclonal Antibody (top) or an Estrogen Receptor alpha Rabbit Monoclonal Antibody (bottom). These two antibodies detect unique, non-overlapping epitopes on human Estrogen Receptor alpha. The similar staining patterns obtained with both antibodies help to confirm the specificity of the staining.
Immunohistochemistry Image 2: Estrogen Receptor alpha (D8H8) Rabbit Monoclonal Antibody
Immunohistochemical analysis of paraffin-embedded normal human breast using Estrogen Receptor alpha (D8H8) Rabbit Monoclonal Antibody.
Immunohistochemistry Image 3: Estrogen Receptor alpha (D8H8) Rabbit Monoclonal Antibody
Immunohistochemical analysis of paraffin-embedded human infiltrating ductal and lobular carcinoma of the breast using Estrogen Receptor alpha (D8H8) Rabbit Monoclonal Antibody.
Immunohistochemistry Image 4: Estrogen Receptor alpha (D8H8) Rabbit Monoclonal Antibody
Orders: 877-616-CELL (2355) [email protected] Support: 877-678-TECH (8324) [email protected] Web: cellsignal.com
For Research Use Only. Not for Use in Diagnostic Procedures.

Revision 13

Cell Signaling Technologyロゴ
Immunohistochemical analysis of paraffin-embedded human endometrioid carcinoma using Estrogen Receptor alpha (D8H8) Rabbit Monoclonal Antibody.
Immunohistochemistry Image 5: Estrogen Receptor alpha (D8H8) Rabbit Monoclonal Antibody
Immunohistochemical analysis of paraffin-embedded human endometrioid carcinoma using Estrogen Receptor alpha (D8H8) Rabbit Monoclonal Antibody.
Immunohistochemistry Image 6: Estrogen Receptor alpha (D8H8) Rabbit Monoclonal Antibody
Immunohistochemical analysis of paraffin-embedded normal human uterus using Estrogen Receptor alpha (D8H8) Rabbit Monoclonal Antibody.
Immunohistochemistry Image 7: Estrogen Receptor alpha (D8H8) Rabbit Monoclonal Antibody
Orders: 877-616-CELL (2355) [email protected] Support: 877-678-TECH (8324) [email protected] Web: cellsignal.com
For Research Use Only. Not for Use in Diagnostic Procedures.

Revision 13

Cell Signaling Technologyロゴ
Chromatin immunoprecipitations were performed with cross-linked chromatin from MCF7 cells grown in phenol red free medium and 5% charcoal stripped FBS for 4 d then treated with β-estradiol (10 nM) for 45 minutes and Estrogen Receptor α (D8H8) Rabbit mAb, using SimpleChIP® Plus Enzymatic Chromatin IP Kit (Magnetic Beads) #9005. DNA Library Prep Kit for Illumina® (ChIP-seq, CUT&RUN) #56795. The figure shows binding across TFF1/pS2, a known target gene of Estrogen Receptor α (see additional figure containing ChIP-qPCR data).
Chromatin Immunoprecipitation Image 1: Estrogen Receptor alpha (D8H8) Rabbit Monoclonal Antibody
Chromatin immunoprecipitations were performed with cross-linked chromatin from MCF7 cells grown in phenol red free medium and 5% charcoal stripped FBS for 4 d then treated with β-estradiol (10 nM) for 45 minutes and 5 μl of Estrogen Receptor α (D8H8) Rabbit mAb, using SimpleChIP® Plus Enzymatic Chromatin IP Kit (Magnetic Beads) #9005. DNA Library Prep Kit for Illumina® (ChIP-seq, CUT&RUN) #56795. The figure shows binding across chromosome 21 (upper), including TFF1/pS2 (lower), a known target gene of Estrogen Receptor α (see additional figure containing ChIP-qPCR data).
Chromatin Immunoprecipitation Image 2: Estrogen Receptor alpha (D8H8) Rabbit Monoclonal Antibody
Chromatin immunoprecipitations were performed with cross-linked chromatin from MCF7 cells grown in phenol red free medium and 5% charcoal stripped FBS for 4 d then treated with β-estradiol (10 nM) for 45 minutes and either Estrogen Receptor α (D8H8) Rabbit mAb or Normal Rabbit IgG #2729 using SimpleChIP® Enzymatic Chromatin IP Kit (Magnetic Beads) #9003. The enriched DNA was quantified by real-time PCR using SimpleChIP® Human ESR1 Promoter Primers #9673, SimpleChIP® Human pS2 Promoter Primers #9702, and SimpleChIP® Human α Satellite Repeat Primers #4486. The amount of immunoprecipitated DNA in each sample is represented as signal relative to the total amount of input chromatin, which is equivalent to one.
Chromatin Immunoprecipitation Image 3: Estrogen Receptor alpha (D8H8) Rabbit Monoclonal Antibody
Orders: 877-616-CELL (2355) [email protected] Support: 877-678-TECH (8324) [email protected] Web: cellsignal.com
For Research Use Only. Not for Use in Diagnostic Procedures.

Revision 13

Cell Signaling Technologyロゴ
CUT&RUN was performed with MCF7 cells grown in phenol red free medium and 5% charcoal stripped FBS for 4 days, then treated with β-estradiol (10 nM) for 45 minutes and either Estrogen Receptor a (D8H8) Rabbit mAb or Estrogen Receptor α (D6R2W) Rabbit mAb #13258, using CUT&RUN Assay Kit #86652. DNA Libraries were prepared using DNA Library Prep Kit for Illumina® #56795. The figure shows binding across TFF1 gene.
CUT & RUN Image 1: Estrogen Receptor alpha (D8H8) Rabbit Monoclonal Antibody
CUT&RUN was performed with MCF7 cells grown in phenol red free medium and 5% charcoal stripped FBS for 4 days, then treated with β-estradiol (10 nM) for 45 minutes and either Estrogen Receptor a (D8H8) Rabbit mAb or Estrogen Receptor α (D6R2W) Rabbit mAb #13258, using CUT&RUN Assay Kit #86652. DNA Libraries were prepared using DNA Library Prep Kit for Illumina® #56795. The figures show binding across chromosome 21 (upper), including TFF1 gene (lower).
CUT & RUN Image 2: Estrogen Receptor alpha (D8H8) Rabbit Monoclonal Antibody
CUT&RUN was performed with MCF7 cells grown in phenol red free medium and 5% charcoal stripped FBS for 4 days, then treated with β-estradiol (10 nM) for 45 minutes and either Estrogen Receptor a (D8H8) Rabbit mAb or Rabbit (DA1E) mAb IgG XP® Isotype Control (CUT&RUN) #66362, using CUT&RUN Assay Kit #86652. The enriched DNA was quantified by real-time PCR using SimpleChIP® Human PS2 Promoter Primers #9702 and SimpleChIP® Human α Satellite Repeat Primers #4486. The amount of immunoprecipitated DNA in each sample is represented as signal relative to the total amount of input chromatin, which is equivalent to one.
CUT & RUN Image 3: Estrogen Receptor alpha (D8H8) Rabbit Monoclonal Antibody
Orders: 877-616-CELL (2355) [email protected] Support: 877-678-TECH (8324) [email protected] Web: cellsignal.com
For Research Use Only. Not for Use in Diagnostic Procedures.

Revision 13

Cell Signaling Technologyロゴ
CUT&Tag was performed with MCF7 cells grown in phenol red free medium and 5% charcoal stripped FBS for 4 days, then treated with β-estradiol (10nM) for 45 minutes and Estrogen Receptor alpha (D8H8) Rabbit mAb, using CUT&Tag Assay Kit #77552. DNA library was prepared using CUT&Tag Dual Index Primers and PCR Master Mix for Illumina Systems #47415. The figures show binding across chromosome 11 (upper), including the CCND1 gene (lower).
CUT & Tag Image 1: Estrogen Receptor alpha (D8H8) Rabbit Monoclonal Antibody
CUT&Tag was performed with MCF7 cells grown in phenol red free medium and 5% charcoal stripped FBS for 4 days, then treated with β-estradiol (10nM) for 45 minutes and Estrogen Receptor alpha (D8H8) Rabbit mAb, using CUT&Tag Assay Kit #77552. DNA library was prepared using CUT&Tag Dual Index Primers and PCR Master Mix for Illumina Systems #47415. The figure shows binding across the CCND1 gene.
CUT & Tag Image 2: Estrogen Receptor alpha (D8H8) Rabbit Monoclonal Antibody
Orders: 877-616-CELL (2355) [email protected] Support: 877-678-TECH (8324) [email protected] Web: cellsignal.com
For Research Use Only. Not for Use in Diagnostic Procedures.