Recombinant Anti-COX2 / Cyclooxygenase 2 antibody [RM1026] (ab283574)

False colour image of Western blot: Anti-COX2 / Cyclooxygenase 2 antibody [RM1026] 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, ab283574 was shown to bind specifically to COX2 / Cyclooxygenase 2. A band was observed at 74-76 kDa in wild-type A549 cell lysates with no signal observed at this size in PTGS2 knockout cell line ab280802 (knockout cell lysate ab283825). Band at 70 kDa in both wild-type and knockout samples is non-specific but exact protein is not determined. To generate this image, wild-type and PTGS2 knockout A549 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.

Negative control: MCF7 (PMID: 24325753, PMID: 16997132)

Lysates/proteins at 20 µg per lane.

Performed under reducing conditions.

False colour image of Western blot: Anti- COX2 / Cyclooxygenase 2 antibody [RM1026] (ab283574) 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, ab283574 was shown to bind specifically to COX2 / Cyclooxygenase 2. Target band was observed at 74 kDa in wild-type A549 cell lysates with no signal observed at this size in COX2 / Cyclooxygenase 2 knockout cell line ab280802. To generate this image, wild-type and COX2 / Cyclooxygenase 2 knockout A549 cell lysates were analyzed. 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 (IRDye^® 800CW) preabsorbed (ab216773) and Goat anti-Mouse IgG H&L (IRDye^® 680RD) preabsorbed (ab216776) at 1/20000 dilution.

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

Negative control: MCF7 (PMID: 24325753, PMID: 16997132)

Lower bands could be COX-2 fragments due to proteolysis. (PMID: 32366045)

Exposure time: Lane 1-4: 2 min Lane 5-6: 3 min

Immunohistochemical analysis of paraffin-embedded human colon tissue labelling COX2 / Cyclooxygenase 2 with ab283574 at 1/500 dilution (1.058 µg/ml) followed by a ready to use Goat Anti-Rabbit IgG H&L (HRP polymer). Positive staining on human colon. The section was incubated with ab283574 overnight at 4°C. Counterstained with Hematoxylin.

Secondary antibody only control: Secondary antibody is a ready to use Goat Anti-Rabbit IgG H&L (HRP polymer).

Heat mediated antigen retrieval using ab93684 (Tris/EDTA buffer, pH 9.0)

Immunohistochemical analysis of paraffin-embedded human colon carcinoma tissue labelling COX2 / Cyclooxygenase 2 with ab283574 at 1/500 dilution (1.058 µg/ml) followed by a ready to use Goat Anti-Rabbit IgG H&L (HRP polymer). Positive staining on human colon carcinoma. The section was incubated with ab283574 overnight at 4°C. Counterstained with Hematoxylin.

Secondary antibody only control: Secondary antibody is a ready to use Goat Anti-Rabbit IgG H&L (HRP polymer).

Heat mediated antigen retrieval using ab93684 (Tris/EDTA buffer, pH 9.0)

Immunohistochemical analysis of paraffin-embedded human liver tissue labeling COX2 / Cyclooxygenase 2 with ab283574 at 1/500 dilution (1.058 µg/ml) followed by a ready to use Goat Anti-Rabbit IgG H&L (HRP polymer). Positive staining on human colon liver. The section was incubated with ab283574 overnight at 4°C. Counterstained with Hematoxylin.

Secondary antibody only control: Secondary antibody is a ready to use Goat Anti-Rabbit IgG H&L (HRP polymer).

Heat mediated antigen retrieval using ab93684 (Tris/EDTA buffer, pH 9.0)

Immunofluorescent analysis of 4% paraformaldehyde-fixed, 0.1% TritonX-100 permeabilized RAW 264.7 (mouse Abelson murine leukemia virus-induced tumor macrophage) cells labelling COX2 / Cyclooxygenase 2 with ab283574 at 1/1000 dilution (0.529 µg/ml), followed by ab150081 Goat Anti-Rabbit IgG H&L (Alexa Fluor^® 488) preadsorbed antibody at 1/1000 dilution (2 µg/ml)(Green). Confocal image showing cytoplasmic staining in RAW 264.7 cell line after treatment with lipopolysaccharide (1 µg/ml) for 6 hours is observed. ab195889 Anti-alpha Tubulin mouse monoclonal antibody - Microtubule Marker (Alexa Fluor^® 594) was used to counterstain tubulin at 1/200 dilution (2.5 µg/ml)(Red). The Nuclear counterstain was DAPI (Blue).

Secondary antibody only control: Secondary antibody is ab150081 Goat Anti-Rabbit IgG H&L (Alexa Fluor^® 488) preadsorbed at 1/1000 dilution (2 µg/ml).

Immunofluorescent analysis of 4% paraformaldehyde-fixed, 0.1% TritonX-100 permeabilized U-87 MG (human glioblastoma-astrocytoma epithelial cell) cells labelling COX2 / Cyclooxygenase 2 with ab283574 at 1/500 dilution (1.058 µg/ml), followed by ab150081 Goat Anti-Rabbit IgG H&L (Alexa Fluor^® 488) preadsorbed antibody at 1/1000 dilution (2 µg/ml)(Green). Confocal image showing cytoplasmic staining in of U-87 MG cell line. Negative control: MCF7 (PMID:18199541) is observed. ab195889 Anti-alpha Tubulin mouse monoclonal antibody - Microtubule Marker (Alexa Fluor^® 594) was used to counterstain tubulin at 1/200 dilution (2.5 µg/ml)(Red). The Nuclear counterstain was DAPI (Blue).

Secondary antibody only control: Secondary antibody is ab150081 Goat Anti-Rabbit IgG H&L (Alexa Fluor® 488) preadsorbed at 1/1000 dilution (2 µg/ml).

Flow cytometric analysis of 4% paraformaldehyde fixed 90% methanol permeabilized RAW 264.7 (mouse Abelson murine leukemia virus-induced tumor macrophage) treated with 1 µg/ml LPS for 6h (Red) / Untreated control (Green) cells labelling COX2 / Cyclooxygenase 2 with ab283574 at 1/500 dilution (0.1µg) compared with a Rabbit monoclonal IgG (ab172730) (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, ab150081) at 1/500 dilution was used as the secondary antibody.

Flow cytometric analysis of 4% paraformaldehyde fixed 90% methanol permeabilized MCF7 (human breast adenocarcinoma epithelial cell)(Left) / U-87 MG (human glioblastoma-astrocytoma epithelial cell)(Right) cells labelling COX2 / Cyclooxygenase 2 with ab283574 at 1/50 dilution (1µg) (Red) compared with a Rabbit monoclonal IgG (ab172730) (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, ab150081) at 1/500 dilution was used as the secondary antibody.

COX2 / Cyclooxygenase 2 was immunoprecipitated from 0.35 mg U-87 MG (human glioblastoma-astrocytoma epithelial cell) whole cell lysate with ab283574 at 1/30 dilution (2µg in 0.35mg lysates). Western blot was performed on the immunoprecipitate using ab283574 at 1/1000 dilution. VeriBlot for IP secondary antibody(HRP)(ab131366) was used at 1/5000 dilution.

Lane 1: U-87 MG (Human glioblastoma-astrocytoma epithelial cell) whole cell lysate 10µg

Lane 2: ab283574 IP in U-87 MG whole cell lysate

Lane 3:Rabbit monoclonal IgG (ab172730) instead of ab283574 in U-87 MG whole cell lysate

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

Exposure time: 6 seconds

Lower bands could be COX-2 fragments due to proteolysis. (PMID: 32366045)

COX2 / Cyclooxygenase 2 was immunoprecipitated from 0.35 mg RAW 264.7 (mouse Abelson murine leukemia virus-induced tumor macrophage) treated with 1 µg/ml LPS for 6h whole cell lysate with ab283574 at 1/30 dilution (2µg in 0.35mg lysates). Western blot was performed on the immunoprecipitate using ab283574 at 1/1000 dilution. VeriBlot for IP secondary antibody(HRP)(ab131366) was used at 1/5000 dilution.

Lane 1: RAW 264.7 (Mouse Abelson murine leukemia virus-induced tumor macrophage) treated with 1ug/ml LPS for 6h whole cell lysate 10µg

Lane 2: ab283574 IP in RAW 264.7 treated with 1 µg/ml LPS for 6h whole cell lysate

Lane 3: Rabbit monoclonal IgG (ab172730) instead of ab283574 in RAW 264.7 treated with 1 µg/ml LPS for 6h whole cell lysate

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

Exposure time: 6 seconds