Anti-COX2 / Cyclooxygenase 2 antibody [RM1026]

• IHC-P

Supplier Data

Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-COX2 / Cyclooxygenase 2 antibody [RM1026] (AB283574)

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)

• IHC-P

Supplier Data

Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-COX2 / Cyclooxygenase 2 antibody [RM1026] (AB283574)

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)

• IHC-P

Supplier Data

Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-COX2 / Cyclooxygenase 2 antibody [RM1026] (AB283574)

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)

• ICC/IF

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Immunocytochemistry/ Immunofluorescence - Anti-COX2 / Cyclooxygenase 2 antibody [RM1026] (AB283574)

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).

• IP

Supplier Data

Immunoprecipitation - Anti-COX2 / Cyclooxygenase 2 antibody [RM1026] (AB283574)

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)

All lanes:

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

Predicted band size: 69 kDa

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• ICC/IF

Supplier Data

Immunocytochemistry/ Immunofluorescence - Anti-COX2 / Cyclooxygenase 2 antibody [RM1026] (AB283574)

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).

• IP

Supplier Data

Immunoprecipitation - Anti-COX2 / Cyclooxygenase 2 antibody [RM1026] (AB283574)

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

All lanes:

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

Predicted band size: 69 kDa

false

• WB

Lab

Western blot - 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.

All lanes:

Western blot - Anti-COX2 / Cyclooxygenase 2 antibody [RM1026] (ab283574) at 1/1000 dilution

Lane 1:

Wild-type A549 cell lysate at 20 µg

Lane 2:

PTGS2 knockout A549 cell lysate at 20 µg

Lane 3:

U-87 MG cell lysate at 20 µg

Lane 4:

MOLT-4 cell lysate at 20 µg

Observed band size: 74-76 kDa

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• WB

Lab

Western blot - Anti-COX2 / Cyclooxygenase 2 antibody [RM1026] (AB283574)

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

All lanes:

Western blot - Anti-COX2 / Cyclooxygenase 2 antibody [RM1026] (ab283574) at 1/1000 dilution

Lane 1:

U-87 MG (Human glioblastoma-astrocytoma epithelial cell) whole cell lysate at 20 µg

Lane 2:

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

Lane 3:

RAW 264.7 (Mouse Abelson murine leukemia virus-induced tumor macrophage) whole cell lysate at 20 µg

Lane 4:

RAW 264.7 treated with 1 µg/ml lipopolysaccharide (LPS) for 6h whole cell lysate at 20 µg

Lane 5:

C6 (Rat glial tumor glial cell) whole cell lysate at 20 µg

Lane 6:

C6 treated with 100 ng/ml lipopolysaccharide (LPS) for 4h whole cell 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 1/100000 dilution

Predicted band size: 69 kDa

false

• WB

Lab

Western blot - Anti-COX2 / Cyclooxygenase 2 antibody [RM1026] (AB283574)

Compared with ab179800, ab283574 has higher sensitivity, we recommend ab283574 as an alternative for testing COX2 in western blot.

ab181602 was used as a loading control at a 1/1000000 dilution.

Blocking and dilution buffer : 5% NFDM/TBST.

Lanes 1 - 3:

Western blot - Anti-COX2 / Cyclooxygenase 2 antibody [EPR12012] (<a href='/en-us/products/primary-antibodies/cox2-cyclooxygenase-2-antibody-epr12012-ab179800'>ab179800</a>) at 1/1000 dilution

Lanes 4 - 6:

Western blot - Anti-COX2 / Cyclooxygenase 2 antibody [RM1026] (ab283574) at 1/1000 dilution

Lanes 1 and 3:

Wild-type HeLa (human cervix adenocarcinoma epithelial cell) whole cell lysate at 20 µg

Lanes 2 and 4:

PTGS2 knockout HeLa (human cervix adenocarcinoma epithelial cell) whole cell lysate at 20 µg

Lanes 3 and 6:

A549 (Human lung carcinoma epithelial cell) whole cell 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: 69 kDa

Observed band size: 75 kDa

false

Exposure time: 180s

• WB

Supplier Data

Western blot - Anti-COX2 / Cyclooxygenase 2 antibody [RM1026] (AB283574)

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.

All lanes:

Western blot - Anti-COX2 / Cyclooxygenase 2 antibody [RM1026] (ab283574) at 1/10000 dilution

Lane 1:

PTGS2 (COX2 / Cyclooxygenase 2) KO A549 (Human lung carcinoma epithelial cell) cell lysate at 20 µg

Lane 2:

Wild-type A549 cell lysate at 20 µg

Lane 3:

U-87 MG (Human glioblastoma-astrocytoma epithelial cell) whole cell lysate at 20 µg

Lane 4:

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

Secondary

All lanes:

Western blot - Goat anti-Rabbit IgG H&L (IRDye® 800CW) preadsorbed (<a href='/en-us/products/secondary-antibodies/goat-rabbit-igg-h-l-irdye-800cw-preadsorbed-ab216773'>ab216773</a>) at 1/10000 dilution

Predicted band size: 69 kDa

Observed band size: 74 kDa

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