Anti-Estrogen Receptor alpha antibody [E115] - ChIP Grade

22 product images

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  Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-Estrogen Receptor alpha antibody [E115] - ChIP Grade (ab32063)

  Immunohistochemical analysis of formalin fixed paraffin embedded human breast carcinoma labelling Estrogen Receptor alpha with ab32063 at a concentration of 0.5µg/ml. The immunostaining was performed on a Ventana DISCOVERY ULTRA (Roche Tissue Diagnostics) instrument with a OptiView DAB IHC Detection Kit. Heat mediated antigen retrieval was performed with DISCOVERY cell conditioning solution (CC1) 100°C, pH8.5 for 32mins.

  Anti-Estrogen Receptor alpha antibody [E115] ab32063 was incubated for 16mins at 37°C. Sections were counterstained with Hematoxylin II. Image inset shows absence of staining in secondary antibody only control.

  Customers are encouraged to optimise antigen retrieval conditions, antibody concentration, incubation times and temperature for best results in their own IHC assay workflow (automated and manual).

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  This image is courtesy of an anonymous customer review.

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

  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.

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

  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'

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  Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-Estrogen Receptor alpha antibody [E115] - ChIP Grade (ab32063)

  Immunohistochemical staining of paraffin embedded human endometrial carcinoma with purified ab32063 at a working dilution of 1 in 200. The secondary antibody used is Goat Anti-Rabbit IgG H&L (HRP) 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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  Flow Cytometry (Intracellular) - Anti-Estrogen Receptor alpha antibody [E115] - ChIP Grade (ab32063)

  Estrogen Receptor alpha Flow Cytometry (Intracellular) staining using rabbit Anti-Estrogen Receptor alpha antibody

  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) (Goat Anti-Rabbit IgG H&L (Alexa Fluor® 488) 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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  Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-Estrogen Receptor alpha antibody [E115] - ChIP Grade (ab32063)

  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 Antigen Retrieval Buffer (100X Tris-EDTA Buffer, pH 9.0) ab93684 (Tris/EDTA buffer, pH 9.0).
  

  Nuclear staining on human endometrium.

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  Immunocytochemistry/ Immunofluorescence - Anti-Estrogen Receptor alpha antibody [E115] - ChIP Grade (ab32063)

  Estrogen Receptor alpha Immunocytochemistry/ Immunofluorescence staining using rabbit Anti-Estrogen Receptor alpha antibody

  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. Goat Anti-Rabbit IgG H&L (Alexa Fluor® 488) ab150077, an Alexa Fluor^® 488-conjugated goat anti-rabbit IgG (1/1000) was used as the secondary antibody. The cells were co-stained with Anti-alpha Tubulin antibody [DM1A] - Loading Control ab7291, a mouse anti-tubulin (1/1000) using Goat Anti-Mouse IgG H&L (Alexa Fluor® 594) preadsorbed 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, Goat Anti-Mouse IgG H&L (Alexa Fluor® 594) preadsorbed ab150120, an Alexa Fluor^® 594-conjugated goat anti-mouse IgG (1/1000).

  Control 2: Anti-alpha Tubulin antibody [DM1A] - Loading Control ab7291 (1/1000) and secondary antibody, Goat Anti-Rabbit IgG H&L (Alexa Fluor® 488) ab150077, an Alexa Fluor^® 488-conjugated goat anti-rabbit IgG (1/1000).

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  Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-Estrogen Receptor alpha antibody [E115] - ChIP Grade (ab32063)

  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 Antigen Retrieval Buffer (100X Tris-EDTA Buffer, pH 9.0) ab93684 (Tris/EDTA buffer, pH 9.0).
  

  Nuclear staining on human breast carcinoma.

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  Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-Estrogen Receptor alpha antibody [E115] - ChIP Grade (ab32063)

  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 Antigen Retrieval Buffer (100X Tris-EDTA Buffer, pH 9.0) ab93684 (Tris/EDTA buffer, pH 9.0).
  

  Nuclear staining on human breast.

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

  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.

  All lanes: Western blot - Anti-Estrogen Receptor alpha antibody [E115] - ChIP Grade (ab32063) at 1/200 dilution

  Lane 1: Human uterus tissue lysates at 20 µg

  Lane 2: Human kidney tissue lysates at 20 µg

  Lane 3: Human brain tissue lysates at 20 µg

  Lane 4: Mouse uterus tissue lysates at 20 µg

  Lane 5: Mouse ovary tissue lysates at 20 µg

  Lane 6: Mouse kidney tissue lysates at 20 µg

  Lane 7: Mouse brain tissue lysates at 20 µg

  Lane 8: Rat uterus tissue lysates at 20 µg

  Lane 9: Rat ovary tissue lysates at 20 µg

  Secondary

  All lanes: Goat Anti-Rabbit IgG (HRP) with minimal cross-reactivity with human IgG at 1/2000 dilution

  Predicted band size: 66 kDa

  Observed band size: 67 kDa

  Exposure time: 180s

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

  Blocking and diluting buffer: 5% NFDM/TBST.

  All lanes: Western blot - Anti-Estrogen Receptor alpha antibody [E115] - ChIP Grade (ab32063) at 1/1000 dilution

  Lane 1: MCF7 (Human breast adenocarcinoma epithelial cell). Whole cell lysates at 20 µg

  Lane 2: T-47D (human mammary gland ductal carcinoma epithelial cell). Whole cell lysates at 20 µg

  Lane 3: MDA-MB231 (Human breast adenocarcinoma epithelial cell) Whole cell lysates (Negative control) at 20 µg

  Lane 4: HepG2 (Human hepatocellular carcinoma epithelial cell) Whole cell lysates (Negative control) at 20 µg

  Lane 5: Human uterus whole tissue lysate at 20 µg

  Lane 6: Human ovary whole tissue lysate at 20 µg

  Lane 7: Human ovary cancer whole tissue lysate at 20 µg

  Secondary

  All lanes: Western blot - Goat Anti-Rabbit IgG H&L (HRP) (Goat Anti-Rabbit IgG H&L (HRP) ab97051) at 1/20000 dilution

  Predicted band size: 36 kDa, 66 kDa

  Observed band size: 68 kDa

  Exposure time: 50s

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  Immunocytochemistry/ Immunofluorescence - Anti-Estrogen Receptor alpha antibody [E115] - ChIP Grade (ab32063)

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

  Exposure time: 1^st lane: 85 seconds
  2^nd lane: 32 seconds
  

  Blocking and diluting buffer: 5% NFDM/TBST

  All lanes: Western blot - Anti-Estrogen Receptor alpha antibody [E115] - ChIP Grade (ab32063) at 1/1000 dilution

  Lane 1: Rat pituitary whole tissue lysate at 20 µg

  Lane 2: Mouse pituitary whole tissue lysate at 20 µg

  Secondary

  All lanes: Western blot - Goat Anti-Rabbit IgG H&L (HRP) (Goat Anti-Rabbit IgG H&L (HRP) ab97051) at 1/20000 dilution

  Predicted band size: 36 kDa, 66 kDa

  Observed band size: 68 kDa

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

  All lanes: Western blot - Anti-Estrogen Receptor alpha antibody [E115] - ChIP Grade (ab32063) at 1/500 dilution

  All lanes: MCF-7 cell lysate

  Predicted band size: 66 kDa

  Observed band size: 60 kDa

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  Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-Estrogen Receptor alpha antibody [E115] - ChIP Grade (ab32063)

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

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

  Estrogen Receptor alpha Western blot staining using rabbit Anti-Estrogen Receptor alpha antibody

  In Western blot, Anti-GAPDH antibody [EPR16891] - Loading Control (Anti-GAPDH antibody [EPR16891] - Loading Control ab181602) staining at 1/200000 dilution.

  The tissue samples were kindly provided by GemPharmatech. C57BL/6JGpt wildtype mice and Esr1-KO homozygous mice (Strain ID: T052653).

  All lanes: Western blot - Anti-Estrogen Receptor alpha antibody [E115] - ChIP Grade (ab32063) at 1/5000 dilution

  Lane 1: Wild-type mouse uterus tissue lysate (female case1) at 20 µg

  Lane 2: Wild-type mouse uterus tissue lysate (female case2) at 20 µg

  Lane 3: Esr1 knockout mouse uterus tissue lysate (female case1) at 20 µg

  Lane 4: Esr1 knockout mouse uterus tissue lysate (female case2) at 20 µg

  Secondary

  All lanes: Goat Anti-Rabbit IgG (HRP) with minimal cross-reactivity with human IgG at 1/2000 dilution

  Observed band size: 67 kDa

  Exposure time: 180s

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  ChIC/CUT&RUN sequencing - Anti-Estrogen Receptor alpha antibody [E115] - ChIP Grade (ab32063)

  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 Rabbit IgG, monoclonal [EPR25A] - Isotype 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 sequencing - Anti-Estrogen Receptor alpha antibody [E115] - ChIP Grade (ab32063)

  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 Rabbit IgG, monoclonal [EPR25A] - Isotype Control ab172730 is also shown.

  The University of Geneva owns patents relevant to ChIC (Chromatin Immuno-Cleavage) methods.

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  Immunocytochemistry/ Immunofluorescence - Anti-Estrogen Receptor alpha antibody [E115] - ChIP Grade (ab32063)

  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) (Goat Anti-Rabbit IgG H&L (Alexa Fluor® 488) preadsorbed 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) (Alexa Fluor® 594 Anti-alpha Tubulin antibody [DM1A] - Microtubule Marker 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 Goat Anti-Rabbit IgG H&L (Alexa Fluor® 488) preadsorbed ab150081 Goat Anti-Rabbit IgG H&L (Alexa Fluor^® 488) at 1/1000 dilution (2.0 μg/mL).

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  ChIC/CUT&RUN sequencing - Anti-Estrogen Receptor alpha antibody [E115] - ChIP Grade (ab32063)

  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 Rabbit IgG, monoclonal [EPR25A] - Isotype Control ab172730 is also shown.

  The University of Geneva owns patents relevant to ChIC (Chromatin Immuno-Cleavage) methods.

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  Immunocytochemistry/ Immunofluorescence - Anti-Estrogen Receptor alpha antibody [E115] - ChIP Grade (ab32063)

  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) (Goat Anti-Rabbit IgG H&L (Alexa Fluor® 488) preadsorbed 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) (Alexa Fluor® 594 Anti-alpha Tubulin antibody [DM1A] - Microtubule Marker 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 Goat Anti-Rabbit IgG H&L (Alexa Fluor® 488) preadsorbed ab150081 Goat Anti-Rabbit IgG H&L (Alexa Fluor^® 488) at 1/1000 dilution (2.0 μg/mL).

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  Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-Estrogen Receptor alpha antibody [E115] - ChIP Grade (ab32063)

  Estrogen Receptor alpha Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) staining using rabbit Anti-Estrogen Receptor alpha antibody

  Immunohistochemical analysis of paraffin-embedded tissue labeling Estrogen Receptor alpha with ab32063 at 1/10000 dilution, followed by a ready to use LeicaDS9800 (Bond^™ Polymer Refine Detection).

  Positive staining on (A) Uterus tissue from wild-type C57BL/6JGpt mice and no staining on (B) Uterus tissue from Esr1 knockout mice.
  
  The section was incubated with ab32063 for 30 mins at room temperature.
  
  The immunostaining was performed on a Leica Biosystems BOND^® RX instrument.
  
  The tissue samples were kindly provided by GemPharmatech. C57BL/6JGpt wildtype mice and Esr1-KO homozygous mice (Strain ID: T052653).

  Counterstained with Hematoxylin.

  Secondary antibody only control: Secondary antibody is a ready to use LeicaDS9800 (Bond^™ Polymer Refine Detection).

  Heat mediated antigen retrieval with Tris-EDTA buffer (pH 9.0, epitope retrieval solution2) for 20 mins.