Anti-Retinoic Acid Receptor alpha antibody [EPR23871-271]

• IHC-P

Unknown

Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-Retinoic Acid Receptor alpha antibody [EPR23871-271] (AB275745)

Immunohistochemical analysis of paraffin-embedded Human testis tissue labeling Retinoic Acid Receptor alpha with ab275745 at 1/1000 (2.71 ug/ml) dilution followed by a ready to use LeicaDS9800 (Bond™ Polymer Refine Detection) was used. Nuclear staining on sertoli cells of human testis. The section was incubated with ab275745 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 using ab93684 (Tris/EDTA buffer, pH 9.0).

• IP

Unknown

Immunoprecipitation - Anti-Retinoic Acid Receptor alpha antibody [EPR23871-271] (AB275745)

Retinoic Acid Receptor alpha was immunoprecipitated from 0.35 mg HEK-293 (human embryonic kidney epithelial cell) whole cell lysate with ab275745 at 1/30 dilution (2ug in 0.35mg lysates). Western blot was performed on the immunoprecipitate using ab275745 at 1/1000 dilution. VeriBlot for IP Detection Reagent (HRP)(ab131366) was used at 1/5000 dilution.

Lane 1 : HEK-293 (human embryonic kidney epithelial cell) whole cell lysate 10 ug

Lane 2 : ab275745 IP in HEK-293 whole cell lysate

Lane 3 : Rabbit monoclonal IgG (ab172730) instead of ab275745 in HEK-293 whole cell lysate

Blocking and dilution buffer and concentration : 5% NFDM/TBST.

Exposure time : 84 seconds.

All lanes:

Immunoprecipitation - Anti-Retinoic Acid Receptor alpha antibody [EPR23871-271] (ab275745)

Predicted band size: 51 kDa

Observed band size: 52 kDa

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• IHC-P

Unknown

Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-Retinoic Acid Receptor alpha antibody [EPR23871-271] (AB275745)

Immunohistochemical analysis of paraffin-embedded Mouse testis tissue labeling Retinoic Acid Receptor alpha with ab275745 at 1/1000 (0.542 ug/ml) dilution followed by a ready to use LeicaDS9800 (Bond™ Polymer Refine Detection) was used. Nuclear staining on sertoli cells of mouse testis (PMID : 16210368). The section was incubated with ab275745 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) was used. Heat mediated antigen retrieval using ab93684 (Tris/EDTA buffer, pH 9.0).

• IHC-P

Unknown

Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-Retinoic Acid Receptor alpha antibody [EPR23871-271] (AB275745)

Immunohistochemical analysis of paraffin-embedded Rat testis tissue labeling Retinoic Acid Receptor alpha with ab275745 at 1/1000 (0.542 ug/ml) dilution followed by a ready to use LeicaDS9800 (Bond™ Polymer Refine Detection) was used. Nuclear staining on sertoli cells of rat testis. The section was incubated with ab275745 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 using ab93684 (Tris/EDTA buffer, pH 9.0).

• IP

Unknown

Immunoprecipitation - Anti-Retinoic Acid Receptor alpha antibody [EPR23871-271] (AB275745)

Retinoic Acid Receptor alpha was immunoprecipitated from 0.35 mg NIH/3T3 (mouse embryonic fibroblast) whole cell lysate with ab275745 at 1/30 dilution (2ug in 0.35mg lysates). Western blot was performed on the immunoprecipitate using ab275745 at 1/1000 dilution. VeriBlot for IP Detection Reagent (HRP)(ab131366) was used at 1/5000 dilution.

Lane 1 : NIH/3T3 (mouse embryonic fibroblast) whole cell lysate 10 ug

Lane 2 : ab275745 IP in NIH/3T3 whole cell lysate

Lane 3 : Rabbit monoclonal IgG (ab172730) instead of ab275745 in NIH/3T3 whole cell lysate

Blocking and dilution buffer and concentration : 5% NFDM/TBST.

Exposure time : 84 seconds.

All lanes:

Immunoprecipitation - Anti-Retinoic Acid Receptor alpha antibody [EPR23871-271] (ab275745)

Predicted band size: 51 kDa

Observed band size: 52 kDa

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• WB

Lab

Western blot - Anti-Retinoic Acid Receptor alpha antibody [EPR23871-271] (AB275745)

Anti-RARA antibody [EPR23871-271] (ab275745) staining at 1/1000 dilution, shown in green; Mouse anti-GAPDH antibody [6C5] (ab8245) loading control staining at 1/20000 dilution, shown in red. In Western blot, ab275745 was shown to bind specifically to RARA. A band was observed at 50-55 kDa in wild-type MCF7 cell lysates with no signal observed at this size in RARA knockout cell line ab277158 (knockout cell lysate ab281383). To generate this image, wild-type and RARA 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-Retinoic Acid Receptor alpha antibody [EPR23871-271] (ab275745) at 1/1000 dilution

Lane 1:

Wild-type MCF7 cell lysate at 20 µg

Lane 2:

Western blot - Human RARA (Retinoic Acid Receptor alpha) knockout MCF7 cell line (ab277158)

Lane 2:

Western blot - Human RARA (Retinoic Acid Receptor alpha) knockout MCF7 cell lysate (ab281383)

Lane 2:

RARA knockout MCF7 cell lysate at 20 µg

Lane 3:

Human Testis cell lysate at 20 µg

Lane 4:

Human Brain cell lysate at 20 µg

Secondary

All lanes:

Goat anti-Rabbit IgG H&L 800CW and Goat anti-Mouse IgG H&L 680RD at 1/20000 dilution

Predicted band size: 51 kDa

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• WB

Lab

Western blot - Anti-Retinoic Acid Receptor alpha antibody [EPR23871-271] (AB275745)

Blocking and diluting buffer and concentration : 5% NFDM/TBST.

Low expression : MOLT-4.(Database : HPA).

Exposure time : Lane 1 : 8 seconds Lane2-4 : 70 seconds.

All lanes:

Western blot - Anti-Retinoic Acid Receptor alpha antibody [EPR23871-271] (ab275745) at 1/1000 dilution

Lane 1:

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

Lane 2:

U937 (human histiocytic lymphoma monocyte) whole cell lysate at 20 µg

Lane 3:

HEK-293 (human embryonic kidney epithelial cell) whole cell lysate at 20 µg

Lane 4:

MOLT-4 (human lymphoblastic leukemia T lymphoblast) whole cell lysate at 20 µg

Secondary

All lanes:

Goat Anti-Rabbit IgG, (H+L), Peroxidase conjugated (<a href='/en-us/products/secondary-antibodies/goat-rabbit-igg-h-l-hrp-ab97051'>ab97051</a>) at 1/20000 dilution

Predicted band size: 51 kDa

Observed band size: 52 kDa

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• WB

Lab

Western blot - Anti-Retinoic Acid Receptor alpha antibody [EPR23871-271] (AB275745)

Lanes 1 - 4 : Merged signal (red and green). Green - ab275745 observed at 40 kDa. Red - loading control, ab8245 (Mouse anti-GAPDH antibody [6C5]) observed at 37 kDa.

ab275745 was shown to react with Retinoic Acid Receptor alpha in wild-type HeLa cells in western blot. The bands observed in RARA knockout cell line ab265176 (RARA knockout cell lysate ab257629) below 40 kDa may represent truncated forms and cleaved fragments. This has not been investigated further. Wild-type and RARA CRISPR/Cas9 HeLa edited cell lysates were subjected to SDS-PAGE. Membranes were blocked in 3 % milk in TBS-T (0.1 % Tween^®) before incubation with ab275745 and ab8245 (Mouse anti-GAPDH antibody [6C5]) overnight at 4°C at a 1 in 1000 dilution and a 1 in 20000 dilution respectively. Blots were incubated 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 in 20000 dilution for 1 h at room temperature before imaging.

All lanes:

Western blot - Anti-Retinoic Acid Receptor alpha antibody [EPR23871-271] (ab275745) at 1/1000 dilution

Lane 1:

Wild-type HeLa cell lysate at 20 µg

Lane 2:

RARA CRISPR-Cas9 edited HeLa cell lysate at 20 µg

Lane 2:

Western blot - Human RARA (Retinoic Acid Receptor alpha) knockout HeLa cell line (<a href='/en-us/products/cell-lines/human-rara-retinoic-acid-receptor-alpha-knockout-hela-cell-line-ab265176'>ab265176</a>)

Lane 3:

MCF7 cell lysate at 20 µg

Lane 4:

HepG2 cell lysate at 20 µg

Predicted band size: 51 kDa

Observed band size: 50-55 kDa

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• WB

Lab

Western blot - Anti-Retinoic Acid Receptor alpha antibody [EPR23871-271] (AB275745)

Blocking and diluting buffer and concentration : 5% NFDM/TBST.

Low expression : human brain, human skeletal muscle (Database : GTExPortal, HPA).

Exposure time : 103 seconds.

All lanes:

Western blot - Anti-Retinoic Acid Receptor alpha antibody [EPR23871-271] (ab275745) at 1/1000 dilution

Lane 1:

Human testis tissue lysate at 20 µg

Lane 2:

Human brain tissue lysate at 20 µg

Lane 3:

Human skeletal muscle tissue lysate at 20 µg

Secondary

All lanes:

VeriBlot for IP secondary antibody(HRP)(<a href='/en-us/products/reagents/veriblot-for-ip-detection-reagent-hrp-ab131366'>ab131366</a>) at 1/1000 dilution

Predicted band size: 51 kDa

Observed band size: 52 kDa

false

• WB

Lab

Western blot - Anti-Retinoic Acid Receptor alpha antibody [EPR23871-271] (AB275745)

Blocking and diluting buffer and concentration : 5% NFDM/TBST.

Exposure time : 70 seconds.

All lanes:

Western blot - Anti-Retinoic Acid Receptor alpha antibody [EPR23871-271] (ab275745) at 1/1000 dilution

Lane 1:

NIH/3T3 (mouse embryonic fibroblast) whole cell lysate at 20 µg

Lane 2:

PC-12 (rat adrenal gland pheochromocytoma) whole cell lysate at 20 µg

Secondary

All lanes:

Goat Anti-Rabbit IgG, (H+L), Peroxidase conjugated (<a href='/en-us/products/secondary-antibodies/goat-rabbit-igg-h-l-hrp-ab97051'>ab97051</a>) at 1/20000 dilution

Predicted band size: 51 kDa

Observed band size: 52 kDa

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