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Synthetic peptide (the amino acid sequence is considered to be commercially sensitive) within Human Estrogen Receptor alpha. The exact sequence is proprietary.
(Peptide available as
This product is a recombinant rabbit monoclonal antibody.
Alternative versions available:
Anti-Estrogen Receptor alpha antibody (HRP) [E115] (ab194152)
Anti-Estrogen Receptor alpha antibody (Alexa Fluor® 488) [E115] - (ab194150)
Anti-Estrogen Receptor alpha antibody (Phycoerythrin) [E115] (ab209288)
Produced using Abcam's RabMAb® technology. RabMAb® technology is covered by the following U.S. Patents, No. 5, 675, 063 and/or 7, 429, 487.
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.
Our Abpromise guarantee covers the use of ab32063 in the following tested applications.
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).|
For unpurified, use 1/50 - 1/100.
Blocking buffer: 5% NFDM/TBST
Dilution buffer: 5% NFDM/TBST
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.
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).
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.
Located to the 2 intron of TFF1 gene.
Forward: 5' -agtctcctccaacctgacctt-3'
Reverse: 5' -ttccggccatctctcactat-3'
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 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.
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.