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.

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).

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.

Blocking and diluting buffer: 5% NFDM/TBST. 

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

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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