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

• IHC-P

Lab

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).

• IHC-P

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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 ab93684 (Tris/EDTA buffer pH 9.0).

Nuclear staining on human breast.

• IHC-P

Unknown

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 ab93684 (Tris/EDTA buffer pH 9.0).

Nuclear staining on human breast carcinoma.

• IHC-P

Unknown

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 ab93684 (Tris/EDTA buffer pH 9.0).

Nuclear staining on human endometrium.

• IHC-P

Unknown

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

• IHC-P

Unknown

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 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.

• ChIP

AbReview35027****

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.

This image is courtesy of an anonymous Abreview.

• ChIP

Unknown

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'

• IHC-P

Lab

Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-Estrogen Receptor alpha antibody [E115] - ChIP Grade (AB32063)

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 6.0, epitope retrieval solution1) for 20 mins.

• WB

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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

false

• WB

Lab

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) (<a href='/en-us/products/secondary-antibodies/goat-rabbit-igg-h-l-hrp-ab97051'>ab97051</a>) at 1/20000 dilution

Predicted band size: 36 kDa,66 kDa

Observed band size: 68 kDa

false

Exposure time: 50s

• WB

Lab

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

false

Exposure time: 180s

• ChIC/CUT&RUN-seq

Lab

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 ab172730 is also shown.

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

• ChIC/CUT&RUN-seq

Lab

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 ab172730 is also shown.

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

• ChIC/CUT&RUN-seq

Lab

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 ab172730 is also shown.

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

• WB

Lab

Western blot - Anti-Estrogen Receptor alpha antibody [E115] - ChIP Grade (AB32063)

In Western blot, 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

false

Exposure time: 180s

• WB

Lab

Western blot - Anti-Estrogen Receptor alpha antibody [E115] - ChIP Grade (AB32063)

Exposure time :
Lane 1 : 85 seconds
Lane 2 : 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) (<a href='/en-us/products/secondary-antibodies/goat-rabbit-igg-h-l-hrp-ab97051'>ab97051</a>) at 1/20000 dilution

Predicted band size: 36 kDa,66 kDa

Observed band size: 68 kDa

false