Anti-Estrogen Receptor alpha (mutated Y537S + D538G) antibody [EPR28709-35]

• IHC-P

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Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-Estrogen Receptor alpha (mutated Y537S + D538G) antibody [EPR28709-35] (AB316764)

Immunohistochemical analysis of paraffin-embedded (A) HEK-293T (human embryonic kidney epithelial cell) transfected with a human ERα (Y537S, D538G) expression vector containing a His tag. (B) HEK-293T transfected with a human ERα (Y537S) expression vector containing a His tag. (C) HEK-293T transfected with a human ERα (D538G) expression vector containing a His tag. (D) HEK-293T transfected with a human ERα expression vector containing a His tag. (E) HEK-293T transfected with empty vector containing a His tag. tissue labeling Estrogen Receptor alpha (mutated Y537S + D538G) with ab316764 at 1/2000 (0.266 ug/ml) dilution, followed by a ready to use LeicaDS9800 (Bond™ Polymer Refine Detection).

Positive staining on 293T overexpressing (A) human ERα (Y537S, D538G). No staining on 293T overexpressing human (B) ERα (Y537S), (C) ERα (D538G), (D) wild-type ERα, or (E) the empty vector control.
The section was incubated with ab316764 for 30 mins at room temperature.
The immunostaining was performed on a Leica Biosystems BOND® RX instrument

Counterstained with Hematoxylin.

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

Heat mediated antigen retrieval was performed with Tris-EDTA buffer (pH 9.0, Epitope Retrieval Solution2) for 20 mins

• Flow Cyt (Intra)

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Flow Cytometry (Intracellular) - Anti-Estrogen Receptor alpha (mutated Y537S + D538G) antibody [EPR28709-35] (AB316764)

Flow cytometric analysis of 4% paraformaldehyde fixed 90% methanol permeabilized Isotype (Upper Left) / 293T cells transfected with a human ERα (Y537S) expression vector containing a myc-His-tag® (Upper Middle) / 293T cells transfected with a human ERα (D538G) expression vector containing a myc-His-tag® (Upper Right) / 293T cells transfected with a human ERα (Y537S, D538G) expression vector containing a myc-His-tag® (Lower Left) / 293T cells transfected with a human wild type ERα expression vector containing a myc-His-tag® (Lower Middle) / 293T cells transfected with an empty expression vector containing a myc-His-tag® (Lower Right) cells labelling Estrogen Receptor alpha (mutated Y537S + D538G) with ab316764 at 1/5000 dilution (0.01 ug)/Middle, Right and Lower Left compared with a Rabbit monoclonal IgG (ab172730) / Left isotype control.

Goat Anti-Rabbit IgG (Alexa Fluor® 488, ab150081) at 1/5000 dilution was used as the secondary antibody.

Cells are co-stained with Myc tag conjugated to Alexa Fluor®647.

• IP

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Immunoprecipitation - Anti-Estrogen Receptor alpha (mutated Y537S + D538G) antibody [EPR28709-35] (AB316764)

Estrogen Receptor alpha (mutated Y537S + D538G) was immunoprecipitated from 0.35 mg 293T (human embryonic kidney epithelial cell) cells transfected with a human ERα (Y537S, D538G) expression vector containing a myc-His-tag®, whole cell lysate with ab316764 at 1/30 dilution (2ug in 0.35mg lysates). Western blot was performed on the immunoprecipitate using ab316764 at 1/1000 dilution. VeriBlot for IP secondary antibody(HRP)(ab131366) was used at 1/5000 dilution.

Lane 1 : 293T (human embryonic kidney epithelial cell) cells transfected with a human ERα (Y537S, D538G) expression vector containing a myc-His-tag®, whole cell lysate
Lane 2 : ab316764 IP in 293T (human embryonic kidney epithelial cell) cells transfected with a human ERα (Y537S, D538G) expression vector containing a myc-His-tag®, whole cell lysate
Lane 3 : Rabbit monoclonal IgG (ab172730) instead of ab316764 in 293T cells transfected with a human ERα (Y537S, D538G) expression vector containing a myc-His-tag®, whole cell lysate

All lanes:

Immunoprecipitation - Anti-Estrogen Receptor alpha (mutated Y537S + D538G) antibody [EPR28709-35] (ab316764) at 1/30 dilution

All lanes:

293T (human embryonic kidney epithelial cell) cells transfected with a human ERα (Y537S, D538G) expression vector containing a myc-His-tag®, whole cell lysate with 5% NFDM/TBST

Secondary

All lanes:

Immunoprecipitation - VeriBlot for IP Detection Reagent (HRP) (<a href='/en-us/products/reagents/veriblot-for-ip-detection-reagent-hrp-ab131366'>ab131366</a>) at 1/5000 dilution

false

Exposure time: 145s

• WB

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Western blot - Anti-Estrogen Receptor alpha (mutated Y537S + D538G) antibody [EPR28709-35] (AB316764)

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

In Western blot, Anti-6X His tag® antibody [EPR20547] - ChIP Grade (ab213204) staining at 1/5000 dilution.

All lanes:

Western blot - Anti-Estrogen Receptor alpha (mutated Y537S + D538G) antibody [EPR28709-35] (ab316764) at 1/1000 dilution

Lane 1:

293T (human embryonic kidney epithelial cell) cells transfected with an empty vector containing a His-tag, whole cell lysate at 20 µg with 5% NFDM/TBST

Lane 2:

293T cells transfected with human ERα (Y537S) expression vector containing a myc-His-tag®, whole cell lysate at 20 µg with 5% NFDM/TBST

Lane 3:

293T cells transfected with human ERα (D538G) expression vector containing a myc-His-tag®, whole cell lysate at 20 µg with 5% NFDM/TBST

Lane 4:

293T cells transfected with human ERα (Y537S, D538G) expression vector containing a myc-His-tag®, whole cell lysate at 20 µg with 5% NFDM/TBST

Lane 5:

293T cells transfected with human ERα expression vector containing a myc-His-tag®, whole cell lysate at 20 µg with 5% NFDM/TBST

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 1/100000 dilution

Observed band size: 37-66 kDa,36 kDa

true

Exposure time: 92s