Recombinant Anti-Estrogen Receptor alpha antibody [E115] - ChIP Grade (ab32063)
Key features and details
- Produced recombinantly (animal-free) for high batch-to-batch consistency and long term security of supply
- Rabbit monoclonal [E115] to Estrogen Receptor alpha - ChIP Grade
- Suitable for: ICC/IF, ChIC/CUT&RUN-seq, ChIP, WB, Flow Cyt (Intra), IHC-P
- Reacts with: Mouse, Rat, Human
Related conjugates and formulations
Overview
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Product name
Anti-Estrogen Receptor alpha antibody [E115] - ChIP Grade
See all Estrogen Receptor alpha primary antibodies -
Description
Rabbit monoclonal [E115] to Estrogen Receptor alpha - ChIP Grade -
Host species
Rabbit -
Specificity
Expression levels of ER alpha protein vary with sample type. This antibody is unsuitable to test ovary and the tissues with low expression level of Estrogen Receptor alpha, such as kidney, lung and brain, in western blot. And it failed to show good IHC signal on mouse and rat tissue sections when using our IHC testing conditions. For our in-house testing we tested the antibody on a mouse tissue array (breast, spleen, lung, stomach, muscle, pancreas, liver, colon, brain, kidney).
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Tested applications
Suitable for: ICC/IF, ChIC/CUT&RUN-seq, ChIP, WB, Flow Cyt (Intra), IHC-Pmore details -
Species reactivity
Reacts with: Mouse, Rat, Human -
Immunogen
Synthetic peptide. This information is proprietary to Abcam and/or its suppliers.
(Peptide available asab203371) -
Positive control
- WB: MCF-7 and T47-D cell lysates; Human uterus and ovary cancer tissue lysates; Mouse and rat uterus lysates; Rat and mouse pituitary whole tissue lysates. IHC-P: Human breast carcinoma and endometrial carcinoma tissues; human endometrium and breast tissues. ICC/IF: MCF-7 cells, 4T1 cells and GH3 cells. Flow Cyt (intra): MCF-7 cells. ChIP: Chromatin prepared from MCF-7+ß-estraiol 30 min cells. ChIC/CUT&RUN: MCF7 cells.
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General notes
This product is a recombinant monoclonal antibody, which offers several advantages including:
- - High batch-to-batch consistency and reproducibility
- - Improved sensitivity and specificity
- - Long-term security of supply
- - Animal-free production
Our RabMAb® technology is a patented hybridoma-based technology for making rabbit monoclonal antibodies. For details on our patents, please refer to RabMAb® patents.
Properties
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Form
Liquid -
Storage instructions
Shipped at 4°C. Store at +4°C short term (1-2 weeks). Upon delivery aliquot. Store at -20°C. Avoid freeze / thaw cycle. -
Storage buffer
pH: 7.20
Preservative: 0.01% Sodium azide
Constituents: 59% PBS, 40% Glycerol, 0.05% BSA -
Concentration information loading...
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Purity
Protein A purified -
Clonality
Monoclonal -
Clone number
E115 -
Isotype
IgG -
Research areas
Associated products
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Alternative Versions
- Anti-Estrogen Receptor alpha antibody [E115] - Low endotoxin, Azide free (ab167611)
- Alexa Fluor® 488 Anti-Estrogen Receptor alpha antibody [E115] (ab194150)
- HRP Anti-Estrogen Receptor alpha antibody [E115] (ab194152)
- PE Anti-Estrogen Receptor alpha antibody [E115] (ab209288)
- Anti-Estrogen Receptor alpha antibody [E115] - BSA and Azide free (ab271827)
- Alexa Fluor® 568 Anti-Estrogen Receptor alpha antibody [E115] - ChIP Grade (ab312920)
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Assay kits
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ChIP Related Products
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Compatible Secondaries
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Conjugation kits
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Isotype control
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Recombinant Protein
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Related Products
Applications
The Abpromise guarantee
Our Abpromise guarantee covers the use of ab32063 in the following tested applications.
The application notes include recommended starting dilutions; optimal dilutions/concentrations should be determined by the end user.
Application | Abreviews | Notes |
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ICC/IF |
1/200.
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ChIC/CUT&RUN-seq |
Use at an assay dependent concentration.
5 µg |
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ChIP | (1) |
Use 4 µg for 25 µg of chromatin.
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WB | (5) |
1/1000. Detects a band of approximately 60 kDa (predicted molecular weight: 67 kDa).
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Flow Cyt (Intra) |
1/1000.
ab172730 - Rabbit monoclonal IgG, is suitable for use as an isotype control with this antibody. We recommend to use a 30 min blocking step(1XPBS/10%goat serum/0,3M Glycin). |
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IHC-P | (11) |
1/200 - 1/5000. Perform heat mediated antigen retrieval with Tris/EDTA buffer pH 9.0 before commencing with IHC staining protocol.
For unpurified, use 1/50 - 1/100. The antibody failed to show good IHC signal on mouse and rat tissue sections when applied using our IHC testing conditions. |
Notes |
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ICC/IF
1/200. |
ChIC/CUT&RUN-seq
Use at an assay dependent concentration. 5 µg |
ChIP
Use 4 µg for 25 µg of chromatin. |
WB
1/1000. Detects a band of approximately 60 kDa (predicted molecular weight: 67 kDa). |
Flow Cyt (Intra)
1/1000. ab172730 - Rabbit monoclonal IgG, is suitable for use as an isotype control with this antibody. We recommend to use a 30 min blocking step(1XPBS/10%goat serum/0,3M Glycin). |
IHC-P
1/200 - 1/5000. Perform heat mediated antigen retrieval with Tris/EDTA buffer pH 9.0 before commencing with IHC staining protocol. For unpurified, use 1/50 - 1/100. The antibody failed to show good IHC signal on mouse and rat tissue sections when applied using our IHC testing conditions. |
Target
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Function
Nuclear hormone receptor. The steroid hormones and their receptors are involved in the regulation of eukaryotic gene expression and affect cellular proliferation and differentiation in target tissues. Can activate the transcriptional activity of TFF1. -
Sequence similarities
Belongs to the nuclear hormone receptor family. NR3 subfamily.
Contains 1 nuclear receptor DNA-binding domain. -
Domain
Composed of three domains: a modulating N-terminal domain, a DNA-binding domain and a C-terminal ligand-binding domain. -
Post-translational
modificationsPhosphorylated by cyclin A/CDK2. Phosphorylation probably enhances transcriptional activity.
Glycosylated; contains N-acetylglucosamine, probably O-linked.
Ubiquitinated. Deubiquitinated by OTUB1.
Dimethylated by PRMT1 at Arg-260. The methylation may favor cytoplasmic localization.
Palmitoylated (isoform 3). Not biotinylated (isoform 3). -
Cellular localization
Nucleus. Cytoplasm. Cell membrane. A minor fraction is associated with the inner membrane and Nucleus. Cytoplasm. Cell membrane. Associated with the inner membrane via palmitoylation. - Information by UniProt
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Database links
- Entrez Gene: 2099 Human
- Entrez Gene: 13982 Mouse
- Entrez Gene: 24890 Rat
- Omim: 133430 Human
- SwissProt: P03372 Human
- SwissProt: P19785 Mouse
- SwissProt: P06211 Rat
- Unigene: 208124 Human
see all -
Alternative names
- 7*/654 isoform antibody
- 7*/819 2 isoform antibody
- 7*/822 isoform antibody
see all
Images
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Immunofluorescent analysis of 4% paraformaldehyde-fixed, 0.1% Triton X-100 permeabilized 4T1 (mouse mammary gland carcinoma epithelial cell line) cells labelling Estrogen Receptor alpha with primary antibody anti-Estrogen Receptor alpha (ab32063) at 1/200 dilution, followed by Goat Anti-Rabbit IgG H&L (Alexa Fluor® 488) (ab150081) secondary antibody at 1/1000 dilution (2.0 µg/mL). Confocal image showing cytoplasmic and nuclear staining in 4T1 cells. Anti-alpha Tubulin mouse monoclonal antibody - Microtubule Marker (Alexa Fluor® 594) (ab195889) was used to counterstain tubulin at 1/200 dilution (2.5 µg/mL). The nuclear counter stain is DAPI (blue).
The secondary antibody only control is : Secondary antibody is ab150081 Goat Anti-Rabbit IgG H&L (Alexa Fluor® 488) at 1/1000 dilution (2.0 µg/mL).
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Immunofluorescent analysis of 4% paraformaldehyde-fixed, 0.1% Triton X-100 permeabilized GH3 (rat pituitary epithelial cell line) cells labelling Estrogen Receptor alpha with primary antibody anti-Estrogen Receptor alpha (ab32063) at 1/200 dilution, followed by Goat Anti-Rabbit IgG H&L (Alexa Fluor® 488) (ab150081) secondary antibody at 1/1000 dilution (2.0 µg/mL). Confocal image showing cytoplasmic and nuclear staining in GH3 cells. Anti-alpha Tubulin mouse monoclonal antibody - Microtubule Marker (Alexa Fluor® 594) (ab195889) was used to counterstain tubulin at 1/200 dilution (2.5 µg/mL). The nuclear counter stain is DAPI (blue).
The secondary antibody only control is : Secondary antibody is ab150081 Goat Anti-Rabbit IgG H&L (Alexa Fluor® 488) at 1/1000 dilution (2.0 µg/mL).
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Immunocytochemistry/Immunofluorescence analysis of MCF-7 cells labelling Estrogen Receptor alpha with purified ab32063 at 1/1000. Cells were fixed with 4% paraformaldehyde and permeabilized with 0.1% Triton X-100. ab150077, an Alexa Fluor® 488-conjugated goat anti-rabbit IgG (1/1000) was used as the secondary antibody. The cells were co-stained with ab7291, a mouse anti-tubulin (1/1000) using ab150120, an Alexa Fluor® 594-conjugated goat anti-mouse IgG (1/1000) as the secondary antibody. Nuclei counterstained with DAPI (blue).
Control 1: primary antibody (1/1000) and secondary antibody, ab150120, an Alexa Fluor® 594-conjugated goat anti-mouse IgG (1/1000).
Control 2: ab7291 (1/1000) and secondary antibody, ab150077, an Alexa Fluor® 488-conjugated goat anti-rabbit IgG (1/1000).
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Immunohistochemical staining of paraffin embedded human endometrium tissue with ab32063 at a dilution of 1/5000. The secondary antibody used was Goat Anti-Rabbit IgG H&L (HRP Polymer). The sample is counter-stained with hematoxylin. Antigen retrieval was heat mediated using ab93684 (Tris/EDTA buffer, pH 9.0).
Nuclear staining on human endometrium.
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Immunohistochemical staining of paraffin embedded human breast carcinoma tissue with ab32063 at a dilution of 1/5000. The secondary antibody used was Goat Anti-Rabbit IgG H&L (HRP Polymer). The sample is counter-stained with hematoxylin. Antigen retrieval was heat mediated using ab93684 (Tris/EDTA buffer, pH 9.0).
Nuclear staining on human breast carcinoma.
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Immunohistochemical staining of paraffin embedded human breast tissue with ab32063 at a dilution of 1/5000. The secondary antibody used was Goat Anti-Rabbit IgG H&L (HRP Polymer). The sample is counter-stained with hematoxylin. Antigen retrieval was heat mediated using ab93684 (Tris/EDTA buffer, pH 9.0).
Nuclear staining on human breast.
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Immunohistochemical staining of paraffin embedded human endometrial carcinoma with purified ab32063 at a working dilution of 1 in 200. The secondary antibody used is ab97051, a HRP goat anti-rabbit IgG (H+L), at 1/500. The sample is counter-stained with hematoxylin. Antigen retrieval was perfomed using Tris-EDTA buffer, pH 9.0.
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Immunohistochemical analysis of human breast carcinoma using anti-Estrogen Receptor alpha (ab32063, unpurified) diluted 1:50
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All lanes : Anti-Estrogen Receptor alpha antibody [E115] - ChIP Grade (ab32063) at 1/200 dilution
Lane 1 : Human uterus tissue lysates
Lane 2 : Human kidney tissue lysates
Lane 3 : Human brain tissue lysates
Lane 4 : Mouse uterus tissue lysates
Lane 5 : Mouse ovary tissue lysates
Lane 6 : Mouse kidney tissue lysates
Lane 7 : Mouse brain tissue lysates
Lane 8 : Rat uterus tissue lysates
Lane 9 : Rat ovary tissue lysates
Lysates/proteins at 20 µg per lane.
Secondary
All lanes : Goat Anti-Rabbit IgG (HRP) with minimal cross-reactivity with human IgG at 1/2000 dilution
Predicted band size: 67 kDa
Observed band size: 67 kDa
Exposure time: 180 secondsBlocking and diluting buffer: 5% NFDM/TBST.
The expression level of ER66 is high in uterus, moderate in ovary and low in kidney, lung, brain, liver, heart (PMID: 20861365, 24977106, 23675257, 23940668, 22070562), especially low in the tissues from male or young female animals (PMID: 26384003, 23940668). ab32063 is unsuitable to test ovary and the tissues with low expression level of Estrogen Receptor alpha in western blot. -
All lanes : Anti-Estrogen Receptor alpha antibody [E115] - ChIP Grade (ab32063) at 1/1000 dilution
Lane 1 : MCF7 (Human breast adenocarcinoma epithelial cell). Whole cell lysates
Lane 2 : T-47D (human mammary gland ductal carcinoma epithelial cell). Whole cell lysates
Lane 3 : MDA-MB231 (Human breast adenocarcinoma epithelial cell) Whole cell lysates (Negative control)
Lane 4 : HepG2 (Human hepatocellular carcinoma epithelial cell) Whole cell lysates (Negative control)
Lane 5 : Human uterus whole tissue lysate
Lane 6 : Human ovary whole tissue lysate
Lane 7 : Human ovary cancer whole tissue lysate
Lysates/proteins at 20 µg per lane.
Secondary
All lanes : Goat Anti-Rabbit IgG H&L (HRP) (ab97051) at 1/20000 dilution
Predicted band size: 67 kDa
Observed band size: 68 kDa why is the actual band size different from the predicted?
Exposure time: 50 secondsBlocking and diluting buffer: 5% NFDM/TBST.
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ChIC/CUT&RUN was performed using a pAG-MNase at a final concentration of 700 ng/mL, 2.5 x 10^5 MCF7 (Human breast adenocarcinoma epithelial cell) cells treated with phenol red free medium and 5% charcoal stripped FBS for 3 days then treated with β-estradiol (10 nM 45 min) and 5 µg of ab32063 [E115]. The resulting DNA was sequenced on the Illumina NovaSeq 6000 to a depth of 10 million reads. The negative IgG control ab172730 is also shown.
The University of Geneva owns patents relevant to ChIC (Chromatin Immuno-Cleavage) methods.
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ChIC/CUT&RUN was performed using a pAG-MNase at a final concentration of 700 ng/mL, 2.5 x 10^5 MCF7 (Human breast adenocarcinoma epithelial cell) cells treated with phenol red free medium and 5% charcoal stripped FBS for 3 days then treated with β-estradiol (10 nM 45 min) and 5 µg of ab32063 [E115]. The resulting DNA was sequenced on the Illumina NovaSeq 6000 to a depth of 10 million reads. The negative IgG control ab172730 is also shown.
The University of Geneva owns patents relevant to ChIC (Chromatin Immuno-Cleavage) methods.
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ChIC/CUT&RUN was performed using a pAG-MNase at a final concentration of 700 ng/mL, 2.5 x 10^5 MCF7 (Human breast adenocarcinoma epithelial cell) cells treated with phenol red free medium and 5% charcoal stripped FBS for 3 days then treated with β-estradiol (10 nM 45 min) and 5 µg of ab32063 [E115]. The resulting DNA was sequenced on the Illumina NovaSeq 6000 to a depth of 10 million reads. The negative IgG control ab172730 is also shown.
The University of Geneva owns patents relevant to ChIC (Chromatin Immuno-Cleavage) methods.
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ChIP analysis using unpurified ab32063 binding Estrogen Receptor alpha in MCF7 cells derived from Human breast carcinoma. Cells were cross-linked for 10 minutes with 1% formaldehyde. Samples were incubated with undiluted primary antibody for 16 hours at 4°C. Protein binding was detected using real-time PCR.
Positive control: Estrogen treated MCF7 cells tested at PS2 promoter.
Negative Control:IgG ChIP and ethanol-depleted cells tested at PS2 promoter. -
Overlay histogram showing MCF7 cells stained with unpurified ab32063 (red line). The cells were fixed with 4% paraformaldehyde (10 min) and then permeabilized with 0.1% PBS-Tween for 20 min. The cells were then incubated in 1x PBS / 10% normal goat serum / 0.3M glycine to block non-specific protein-protein interactions followed by the antibody (ab32063, 1/1000 dilution) for 30 min at 22°C. The secondary antibody used was Alexa Fluor® 488 goat anti-rabbit IgG (H+L) (ab150077) at 1/2000 dilution for 30 min at 22°C. Isotype control antibody (black line) was rabbit IgG (monoclonal) (1µg/1x106 cells) used under the same conditions. Unlabelled sample (blue line) was also used as a control. Acquisition of >5,000 events were collected using a 20mW Argon ion laser (488nm) and 525/30 bandpass filter. This antibody gave a positive signal in MCF7 cells fixed with 80% methanol (5 min)/permeabilized with 0.1% PBS-Tween for 20 min used under the same conditions.
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All lanes : Anti-Estrogen Receptor alpha antibody [E115] - ChIP Grade (ab32063) at 1/1000 dilution
Lane 1 : Rat pituitary whole tissue lysate
Lane 2 : Mouse pituitary whole tissue lysate
Lysates/proteins at 20 µg per lane.
Secondary
All lanes : Goat Anti-Rabbit IgG H&L (HRP) (ab97051) at 1/20000 dilution
Predicted band size: 67 kDa
Observed band size: 68 kDa why is the actual band size different from the predicted?Exposure time: 1st lane: 85 seconds
2nd lane: 32 secondsBlocking and diluting buffer: 5% NFDM/TBST
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Immunofluorescent staining of MCF7 cells (fixed with 4% PFA and permeablized with TritonX 100) with purified ab32063 at a dilution of 1/250. An Alexa Fluor® 555 goat anti-rabbit antibody was used as the secondary at a dilution of 1/200. The panel on the right shows the DAPI counter-staining.
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Anti-Estrogen Receptor alpha antibody [E115] - ChIP Grade (ab32063) at 1/500 dilution (unpurified) + MCF-7 cell lysate
Predicted band size: 67 kDa
Observed band size: 60 kDa why is the actual band size different from the predicted? -
Chromatin was prepared from MCF-7+β-estraiol 30 min, and MCF-7 cells according to the Abcam X-ChIP protocol. Cells were fixed with formaldehyde for 10 min. The ChIP was performed with 25 μg of chromatin, 4 μg of purified ab32063 (blue), and 20 μLl of anti-rabbit IgG sepharose beads. Rabbit normal IgG was added to the beads control (yellow). The immunoprecipitated DNA was quantified by real time PCR (SYBR approach). Primers are located in the 2nd intron of TFF1 gene.
MCF7 Cells were treated as below:
MCF-7 starved overnight, then treated with 10 nM β-Estradiol in 2% FBS media for 30 min.
Control MCF-7 was starved overnight, then in 2% FBS media for 30 mins.
Primer information:
Located to the 2 intron of TFF1 gene.
Sequence:
Forward: 5' -agtctcctccaacctgacctt-3'
Reverse: 5' -ttccggccatctctcactat-3'
Protocols
To our knowledge, customised protocols are not required for this product. Please try the standard protocols listed below and let us know how you get on.
Datasheets and documents
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SDS download
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Datasheet download
References (143)
ab32063 has been referenced in 143 publications.
- Zhang D et al. Cysteine dioxygenase and taurine are essential for embryo implantation by involving in E2-ERα and P4-PR signaling in mouse. J Anim Sci Biotechnol 14:6 (2023). PubMed: 36604722
- Liu Z et al. The ERα/KDM6B regulatory axis modulates osteogenic differentiation in human mesenchymal stem cells. Bone Res 10:3 (2022). PubMed: 34992221
- Yi M et al. Immunoprecipitation Analyses of Estrogen Receptor α Phosphorylated at Serine 216 in the Mouse Liver. Methods Mol Biol 2418:41-51 (2022). PubMed: 35119658
- Yi C et al. Differential Size Distribution and Estrogen Receptor Cargo of Oviductal Extracellular Vesicles at Various Stages of Estrous Cycle in Mice. Reprod Sci 29:2847-2858 (2022). PubMed: 35137347
- Zhang Y et al. Estrogen Receptor-A in Medial Preoptic Area Contributes to Sex Difference of Mice in Response to Sevoflurane Anesthesia. Neurosci Bull 38:703-719 (2022). PubMed: 35175557