Recombinant 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.

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 CRISPR/Cas9 edited cell line ab265176 (RARA CRISPR/Cas9 edited 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 edited HeLa 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.

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

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

Exposure time: 103 seconds.

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

Exposure time: 70 seconds.

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).

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.

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).

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.

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.

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).