Recombinant
RabMAb

Recombinant Anti-Estrogen Receptor alpha antibody [E115] - ChIP Grade (ab32063)

Overview

  • 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. ab32063 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).
  • Tested applications
    Suitable for: ICC/IF, Flow Cyt, ChIP, WB, IHC-Pmore details
  • Species reactivity
    Reacts with: Mouse, Rat, Human
  • Immunogen

    Synthetic peptide within Human Estrogen Receptor alpha aa 50-150. The exact sequence is proprietary.
    Database link: P03372
    (Peptide available as ab203371)

  • Positive control
    • WB: MCF-7 and T47-D cell lysates. IHC-P: Human breast carcinoma and endometrial carcinoma tissues; human endometrium and breast tissues. ICC/IF: MCF-7 cells. Flow Cyt: MCF-7 cells.
  • General notes

    Our RabMAb® technology is a patented hybridoma-based technology for making rabbit monoclonal antibodies. For details on our patents, please refer to RabMab® patents.

    We are constantly working hard to ensure we provide our customers with best in class antibodies. As a result of this work we are pleased to now offer this antibody in purified format. We are in the process of updating our datasheets. The purified format is designated 'PUR' on our product labels. If you have any questions regarding this update, please contact our Scientific Support team.

    This product is a recombinant rabbit monoclonal antibody.

Properties

Applications

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
ICC/IF 1/200.
Flow Cyt 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).

ChIP Use 4 µg for 25 µg of chromatin.
WB 1/1000. Detects a band of approximately 60 kDa (predicted molecular weight: 67 kDa).Can be blocked with Estrogen Receptor alpha peptide (ab203371).
IHC-P 1/200 - 1/5000.

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

  • 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
    modifications
    Phosphorylated 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
  • Database links
  • Alternative names
    • DKFZp686N23123 antibody
    • ER alpha antibody
    • ER antibody
    • ER-alpha antibody
    • Era antibody
    • ESR antibody
    • ESR1 antibody
    • ESR1_HUMAN antibody
    • ESRA antibody
    • Estradiol receptor antibody
    • Estrogen nuclear receptor alpha antibody
    • Estrogen receptor 1 antibody
    • Estrogen receptor alpha 3*,4,5,6,7*/822 isoform antibody
    • Estrogen receptor alpha antibody
    • Estrogen receptor alpha delta 3*,4,5,6,7*,8*/941 isoform antibody
    • Estrogen receptor alpha delta 3*,4,5,6,7*/819 2 isoform antibody
    • Estrogen receptor alpha delta 4 +49 isoform antibody
    • Estrogen receptor alpha delta 4*,5,6,7*/654 isoform antibody
    • Estrogen receptor alpha delta 4*,5,6,7,8*/901 isoform antibody
    • Estrogen receptor alpha E1 E2 1 2 antibody
    • Estrogen receptor alpha E1 N2 E2 1 2 antibody
    • Estrogen receptor antibody
    • ESTRR antibody
    • NR3A1 antibody
    • Nuclear receptor subfamily 3 group A member 1 antibody
    see all

Images

  • 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.

  • 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).

  • 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 seconds


    Blocking 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.

  • 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.

  • 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.

  • 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 seconds


    Blocking and diluting buffer: 5% NFDM/TBST. 

  • 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.

  • 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.

  • 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 seconds

    Blocking and diluting buffer: 5% NFDM/TBST

     

  • Immunohistochemical analysis of human breast carcinoma using anti-Estrogen Receptor alpha (ab32063, unpurified) diluted 1:50

  • 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.

  • 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'

  • 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?

  • 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.

    See Abreview

References

This product has been referenced in:
  • Zhao Y  et al. Estrogenic Effect of the Extract of QingYan Formula on Reproductive Tissues in Immature Mice. Evid Based Complement Alternat Med 2019:5493714 (2019). Read more (PubMed: 30728846) »
  • Selakovic D  et al. The Impact of Hippocampal Sex Hormones Receptors in Modulation of Depressive-Like Behavior Following Chronic Anabolic Androgenic Steroids and Exercise Protocols in Rats. Front Behav Neurosci 13:19 (2019). Read more (PubMed: 30792631) »
See all 49 Publications for this product

Customer reviews and Q&As

11-20 of 28 Abreviews or Q&A

Application
Western blot
Sample
African Green Monkey Cell lysate - whole cell (COS-7 cells)
Gel Running Conditions
Reduced Denaturing (4-12% Bis-Tris)
Loading amount
100000 cells
Treatment
30 nM Bortezomib for 24 hours
Specification
COS-7 cells
Blocking step
Milk as blocking agent for 1 hour(s) and 0 minute(s) · Concentration: 10% · Temperature: 25°C

Abcam user community

Verified customer

Submitted Jun 26 2015

Application
Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections)
Sample
Horse Tissue sections (vagina)
Antigen retrieval step
Heat mediated - Buffer/Enzyme Used: 10 mM citrate, pH6.0
Permeabilization
No
Specification
vagina
Blocking step
Serum as blocking agent for 1 hour(s) and 0 minute(s) · Concentration: 10% · Temperature: 25°C
Fixative
Formaldehyde

Abcam user community

Verified customer

Submitted Jun 26 2015

Application
Flow Cytometry
Sample
Human Cell (A549 lung cancer cells)
Permeabilization
Yes - 0.2% Triton X-100
Gating Strategy
No gating
Specification
A549 lung cancer cells
Preparation
Cell harvesting/tissue preparation method: trypsin
Sample buffer: Hanks’ Balanced Salt solution (HBSS)
Fixation
Paraformaldehyde

Abcam user community

Verified customer

Submitted Apr 29 2015

Application
Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections)
Blocking step
Serum as blocking agent for 20 minute(s) · Concentration: 2.5% · Temperature: 25°C
Antigen retrieval step
Heat mediated - Buffer/Enzyme Used: citrate buffer pH 6.0
Sample
Mouse Tissue sections (Mouse mammary tumor)
Specification
Mouse mammary tumor
Permeabilization
No
Fixative
Formaldehyde

Abcam user community

Verified customer

Submitted Apr 07 2014

Application
Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections)
Sample
Dog Tissue sections (breast)
Antigen retrieval step
Other
Permeabilization
No
Specification
breast
Blocking step
(agent) for 3 hour(s) and 0 minute(s) · Concentration: 1% · Temperature: 22°C
Fixative
Formalin

Abcam user community

Verified customer

Submitted Sep 30 2013

Application
ChIP
Sample
Human Cell lysate - nuclear (mammary cancer cell line (MCF7))
Specification
mammary cancer cell line (MCF7)
Type
Cross-linking (X-ChIP)
Duration of cross-linking step: 10 minute(s) and 0 second(s)
Specification of the cross-linking agent: 1% Formaldehyde
Detection step
Real-time PCR
Positive control
Estrogen treated cells at PS2 promoter
Negative control
IgG ChIP and estrogen-depleted cells at PS2 promoter

Abcam user community

Verified customer

Submitted Mar 14 2013

Answer

Thank you for your detailed reply. That has been very helpful.

I have a few suggestions to make which may improve the results currently observed, or at least allow us to understand what may be contributing to the problems.

I would firstly try to reduce the non-specific background you have been seeing with the secondary antibodies. This would involve trying the staining again with just the secondary antibodies but optimising the conditions to reduce the non-specific binding you have been seeing. This could involve reducing the concentration of secondary antibody used and the blocking solutions used. I would initially just try with the goat serum at 10% for blocking (as mixing blocking types can lead to increased background) with 0.1% Tween 20. I would also introduce 1% goat serum to the antibody diluents (as well as 0.1% Tween 20). You may also want to consider using a different blocking agent altogether, such as 3% BSA.

If this does not improve the results, you may also want to look into optimising the antigen retrieval used. Too much antigen retrieval can lead to higher background staining.

Another thing to consider is that the ab16813 is of the isoform IgM and I do not think the antibody you are currently using is able to detect this isotype. I would therefore suggest using a secondary antibody specific for mouse IgM.

Once the background has been reduced it may be worthwhile to introduce a separate permiabilisation step when using the primary antibodies to ensure good penetration of the two antibodies into the tissue. You could use 0.2% Triton in PBS for 10 minutes.

I hope this information has been of some help. I would be very interested to hear how you get on.

Until then, I wish you all the best with your research.

Read More

Question

Hello,

As per our talk over the phone, here are the images of the double staining attached.

Tissue collection: The tissues used were rat uterus. When the rat was alive she was perfused with PBS followed by 4% PFA. Tissues collected and stored in the 4% PFA for couple of day and the dehydrated with 20% sucrose solution. The dehydrated tissues crayo-frozen and stored at -80.

Tissue preparation: The frozen tissues were sliced with 10 micro meter thick and fixed on a glass slide by 4% PFA for 10 mins. The washed with PBS (3 times). Antigen retrieved in a microwave for 10 mins then washed with ice cold PBS (2 time). The slided were blocked by 5% NGS and 3% FCS. The primary antibodies in 0.1% trinton in PBS with 1% FCS and 1% NGS. Incubated overnight at 40 C (For confirming the axact dilutions needed we tried 1:100,1:200 and 1:400 and for double staing we used 1:400).

On day two the secondary antibodies were added for ER alpha (Ab32063) FitC goat anti rabbit antibody (1:250) and for ER beta Cy3 goat anti mouse antibody (1:250) was used. Incubated for 90 mins and washed with PBS and then added was Hoescht dye (1:1000). Then washed again with PBS and finally with DM water and mounted with mowiol. Images were captured at 20X and also viewed under 40X oil emmersion (No difference).

Images attached are all for double staining. Wherein I have split the images with different fluorecence and also a double fluoroscence imaged.

My question are,

1. As Estrogen receptors belong to the nuclear receptors and it should stain nucleus largely but both the anti bodies does not show any staining into the nucleus.

2. Also, I see that in my blank slides, I see artifacts of fluorescence which is expected to at the region where I expect protein expression, which is one of the difficult situation to analyse my results. Do you have any suggestion for secondary antibodies?

Awaiting for your reply

Best regards

Read More
Answer

Thank you for sharing that information with me and sorry for the delay in getting back to you. This makes understanding the problems you have encountered easier.

In answer to your questions:

1. The ER alpha and beta would be expected to be located in the nucleus however, it is difficult to tell if you are seeing any specific nuclear staining considering the background staining your are seeing with the secondary antitbodies alone.

2. I would agree that the background you are seeing is making it difficult to interpret the results. I think I'd like to collect a little more information from you in order to be able to advise how to improve the results.

If you could let me know which secondary antibodies you are using (the supplier and catalogue number) this would be helpful. Have these secondary antibodies been used successfully with any other primary antibodies?

Have you performed any staining of any other targets in the tissue?

Which antigen retrieval buffer was used?

How long was the blocking performed for (and at what temperature)?

Was any permiabilization performed? If so, how?

What wash steps were employed between each antibody incubation?

With this information I will hopefully be able to advise as to what may be worth optimising and hopefully reduce the background staining.

I look forward to receiving your reply.

Read More

Question
Answer

Thank you for contacting us. I am sorry this antibody is giving no signal in WB.

Have you validated your transfer for proteins around this MW? I would recommend performing a Ponceau stain to double check that the protein is present on the membrane because the run and transfer conditions you are using are generally more suited to smaller proteins.

It may also help to reduce the concentration of blocking agent in the antibody diluents. Milk should be kept to a concentration of 1% with the primary antibody and removed from the secondary antibody buffer. It is possible that the extra protein may be competing with the antibody and thereby inhibiting the binding.

How long was your exposure time? Have you tried overexposing the blot to see if there are any background bands detected? How long were your wash steps? Washes should be kept to 3x 5 minutes to prevent a reduction of signal.

I hope this helps to improve your results, if not, please let me know and I will be happy to help you further.

Read More

Answer

Thank you for contacting us.

Please check the Abpromise guarantee for these products.

https://www.abcam.com/index.html?pageconfig=abpromise

In double Immunofluorescence both the antibody dilutions should be optimized for best results so you may need to optimize the antibody conditions separately before using them together. Please click the link for more information about double immunostaining;

https://www.abcam.com/index.html?pageconfig=resource&rid=11458

https://www.abcam.com/index.html?pageconfig=resource&rid=11459

Please check the compatible secondary antibodies at the following link;

https://www.abcam.com/index.html?pageconfig=productmap&cl=918

IHC substrates

https://www.abcam.com/index.html?pageconfig=resource&rid=12886

https://www.abcam.com/index.html?pageconfig=resource&rid=12854

I will look forward to receive your order soon.

I hope this information is helpful to you. Please do not hesitate to contact us if you need any more advice or information.

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11-20 of 28 Abreviews or Q&A

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