Anti-Estrogen Related Receptor alpha antibody [EPR46Y] - BSA and Azide free

• Flow Cyt

Supplier Data

Flow Cytometry - Anti-Estrogen Related Receptor alpha antibody [EPR46Y] - BSA and Azide free (AB239879)

This data was developed using ab76228 the same antibody clone in a different buffer formulation.

Flow cytometric analysis of 4% Paraformaldehyde fixed 90% Methanol permeabilized HeLa (Human cervix adenocarcinoma epithelial cell) cells labelling Estrogen Receptor alpha with ab76228 at 1/20 dilution (10 μg/ml) compared with a Rabbit monoclonal IgG (Black) isotype control and an unlabelled control (cells without incubation with primary antibody and secondary antibody) (Blue). A Goat anti rabbit IgG (Alexa Fluor® 488, ab150077) at 1/2000 was used as the secondary antibody.

• IP

Unknown

Immunoprecipitation - Anti-Estrogen Related Receptor alpha antibody [EPR46Y] - BSA and Azide free (AB239879)

ab76228 used at 1 : 20 dilution (2μg) immunoprecipitating Estrogen Related Receptor alpha in Hela whole cell lysates.
Lane 1 (input) : HeLa (Human cervix adenocarcinoma epithelial cell) whole cell lysate 10μg.
Lane 2 (+) : HeLa whole cell lysate.
Lane 3 (-) : Rabbit monoclonal IgG (ab172730) instead of ab190685 in HeLa whole cell lysate.

For western blotting, ab227648 and VeriBlot for IP Detection Reagent (HRP)(ab131366) at 1/1000 dilution were used.

All lanes:

Immunoprecipitation - Anti-Estrogen Related Receptor alpha antibody [EPR46Y] (<a href='/en-us/products/primary-antibodies/estrogen-related-receptor-alpha-antibody-epr46y-ab76228'>ab76228</a>)

Predicted band size: 46 kDa

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• WB

Lab

Western blot - Anti-Estrogen Related Receptor alpha antibody [EPR46Y] - BSA and Azide free (AB239879)

This data was developed using ab76228 the same antibody clone in a different buffer formulation.

All lanes:

Western blot - Anti-Estrogen Related Receptor alpha antibody [EPR46Y] (<a href='/en-us/products/primary-antibodies/estrogen-related-receptor-alpha-antibody-epr46y-ab76228'>ab76228</a>) at 1/1000 dilution

Lane 1:

MCF7 (Human breast adenocarcinoma epithelial cell) whole cell lysate at 20 µg

Lane 2:

Mouse brain lysate at 20 µg

Lane 3:

Mouse kidney lysate at 20 µg

Lane 4:

Rat brain 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: 46 kDa

Observed band size: 55 kDa

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• WB

Lab

Western blot - Anti-Estrogen Related Receptor alpha antibody [EPR46Y] - BSA and Azide free (AB239879)

This data was developed using ab76228 the same antibody clone in a different buffer formulation.

All lanes:

Western blot - Anti-Estrogen Related Receptor alpha antibody [EPR46Y] (<a href='/en-us/products/primary-antibodies/estrogen-related-receptor-alpha-antibody-epr46y-ab76228'>ab76228</a>) at 1/1000 dilution

All lanes:

HeLa (Human cervix adenocarcinoma epithelial cell) whole cell lysate at 15 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: 46 kDa

Observed band size: 55 kDa

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• WB

Unknown

Western blot - Anti-Estrogen Related Receptor alpha antibody [EPR46Y] - BSA and Azide free (AB239879)

This data was developed using ab76228 the same antibody clone in a different buffer formulation.

Lane 1 : Wild-type HAP1 whole cell lysate (20 μg)
Lane 2 : Estrogen Related Receptor alpha knockout HAP1 whole cell lysate (20 μg)
Lane 3 : HeLa whole cell lysate (20 μg)
Lane 4 : MCF7 whole cell lysate (20 μg)

Lanes 1 - 4 : Merged signal (red and green). Green - ab76228 observed at 55 kDa. Red - loading control, ab18058, observed at 130 kDa.

ab76228 was shown to specifically react with Estrogen Related Receptor alpha in wild-type HAP1 cells as signal was lost in Estrogen Related Receptor alpha knockout cells. Wild-type and Estrogen Related Receptor alpha knockout samples were subjected to SDS-PAGE. ab76228 and ab18058 (Mouse anti-Vinculin loading control) were incubated overnight at 4°C at 1/1000 dilution and 1/20000 dilution respectively. Blots were developed with Goat anti-Rabbit IgG H&L (IRDye^® 800CW) preabsorbed ab216773 and Goat anti-Mouse IgG H&L (IRDye^® 680RD) preabsorbed ab216776 secondary antibodies at 1/10000 dilution for 1 hour at room temperature before imaging.

All lanes:

Western blot - Anti-Estrogen Related Receptor alpha antibody [EPR46Y] - BSA and Azide free (ab239879)

Predicted band size: 46 kDa

false

• WB

Lab

Western blot - Anti-Estrogen Related Receptor alpha antibody [EPR46Y] - BSA and Azide free (AB239879)

This data was developed using ab76228, the same antibody clone in a different buffer formulation.

Western blot : Anti-ESRRA antibody [EPR46Y] (ab76228) staining at 1/2000 dilution, shown in green; Mouse anti-CANX [CANX/1543] (ab238078) loading control staining at 1/20000 dilution, shown in magenta. In Western blot, ab76228 was shown to bind specifically to ESRRA. A band was observed at 46 kDa in wild-type MCF7 cell lysates with no signal observed at this size in ESRRA knockout cell line. To generate this image, wild-type and ESRRA knockout MCF7 cell lysates were analysed. First, samples were run on an SDS-PAGE gel then transferred onto a nitrocellulose membrane. Membranes were blocked in 3 % milk in TBS-0.1 % Tween^® 20 (TBS-T) before incubation with primary antibodies overnight at 4 °C. Blots were washed four times in TBS-T, incubated with secondary antibodies for 1 h at room temperature, washed again four times then imaged. Secondary antibodies used were Goat anti-Rabbit IgG H&L 800CW and Goat anti-Mouse IgG H&L 680RD at 1/20000 dilution.

All lanes:

Western blot - Anti-Estrogen Related Receptor alpha antibody [EPR46Y] (<a href='/en-us/products/primary-antibodies/estrogen-related-receptor-alpha-antibody-epr46y-ab76228'>ab76228</a>) at 1/2000 dilution

Lane 1:

Wild-type MCF7 cell lysate at 20 µg

Lane 2:

Western blot - Human ESRRA knockout MCF7 cell line (ab289292)

Lane 2:

ESRRA knockout MCF7 cell lysate at 20 µg

Lane 3:

Wild-type HAP1 cell lysate at 20 µg

Lane 4:

ESRRA knockout HAP1 cell lysate at 20 µg

Secondary

Lanes 1 - 4:

Goat anti-Rabbit IgG H&L 800CW at 1/20000 dilution

Lanes 1 - 4:

Goat anti-Mouse IgG H&L 680RD at 1/20000 dilution

Observed band size: 46 kDa

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