Recombinant Anti-COX2 / Cyclooxygenase 2 antibody [EPR12012] (ab179800)

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 [EPR12012] (ab179800) 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, ab179800 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.

False colour image of Western blot: Anti-COX2 / Cyclooxygenase 2 antibody [EPR12012] 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, ab179800 was shown to bind specifically to COX2 / Cyclooxygenase 2. A band was observed at 75 kDa in wild-type A549 cell lysates with no signal observed at this size in PTGS2 knockout cell line ab280802 (knockout cell lysate ab283825). 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 fluorescent western blot (TBS-based) blocking solution 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.

Lanes 1 - 4: Merged signal (red and green). Green - ab179800 observed at 75 kDa. Red - loading control, ab8245 observed at 37 kDa.

 ab179800 was shown to react with COX2 / Cyclooxygenase 2 in wild-type HeLa cells. Loss of signal was observed when knockout cell line ab255420 (knockout cell lysate ab263795) was used. Wild-type and COX2 / Cyclooxygenase 2 knockout samples were subjected to SDS-PAGE. ab179800 and Anti-GAPDH antibody [6C5] - Loading Control (ab8245) were incubated overnight at 4°C at 1 in 1000 dilution and 1 in 20000 dilution respectively. Blots were developed with Goat anti-Rabbit IgG H&L (IRDye^® 800CW) preadsorbed (ab216773) and Goat anti-Mouse IgG H&L (IRDye^® 680RD) preadsorbed (ab216776) secondary antibodies at 1 in 20000 dilution for 1 hour at room temperature before imaging.

Immunocytochemistry/ Immunofluorescence analysis of U-87 MG (human glioblastoma-astrocytoma epithelial cell) cells labeling COX2 / Cyclooxygenase 2 with ab179800 at 1/50 dilution. ab150077 (AlexaFluor^®488 Goat anti-Rabbit) at 1/1000 was used as secondary antibody. Cells were fixed with 4% Paraformaldehyde and permeabilised with 0.1% TritonX-100. ab195889, Anti-alpha Tubulin antibody [DM1A] - Microtubule Marker (Alexa Fluor^® 594) at 1/200 was used as counterstain. Nuclie were stained blue with DAPI.

Confocal image showing cytoplasmic staining in U-87 MG cell line.
Negative control: MCF7 （PMID: 18199541）

Exposure time

Lane 1: 3.25 seconds
Lane 2 and 3: 180 seconds

The expression profile observed in HCT 116 and MCF7 are consistent with the literatures (PMID: 14739610, PMID: 24325753, PMID: 16997132).
Negative control: HCT 116 (PMID: 14739610) and MCF7 (PMID: 24325753, PMID: 16997132)

Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) analysis of human colonic carcinoma tissue labelling COX2 / Cyclooxygenase 2 with unpurified ab179800 at a dilution of 1/250.

Perform heat mediated antigen retrieval before commencing with IHC staining protocol.

Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded) analysis of human colon tissue labeling COX2 / Cyclooxygenase 2 with unpurified ab179800 at a dilution of 1/250.

Perform heat mediated antigen retrieval before commencing with IHC staining protocol.

Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded) analysis of human liver tissue labelling COX2 / Cyclooxygenase 2 with unpurified ab179800 at a dilution of 1/250.

Perform heat mediated antigen retrieval before commencing with IHC staining protocol.

Blocking and dilution buffer: 5% NFDM/TBST.

Blocking and dilution buffer: 5% NFDM/TBST.

Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) analysis of rat kidney tissue labelling COX2 / Cyclooxygenase 2 with purified ab179800 at 1/100. Heat mediated antigen retrieval was performed using Tris/EDTA buffer pH 9. ab97051, a HRP-conjugated goat anti-rabbit IgG (H+L) was used as the secondary antibody (1/500). Negative control using PBS instead of primary antibody. Counterstained with hematoxylin.

Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) analysis of human lung carcinoma tissue labelling COX2 / Cyclooxygenase 2 with purified ab179800 at 1/100. Heat mediated antigen retrieval was performed using Tris/EDTA buffer pH 9. ab97051, a HRP-conjugated goat anti-rabbit IgG (H+L) was used as the secondary antibody (1/500). Negative control using PBS instead of primary antibody. Counterstained with hematoxylin.

ab179800 (purified) at 1/30 immunoprecipitating COX2 in A549 whole cell lysate.

Lane 1 (input): A549 whole cell lysate (10µg)

Lane 2 (+): ab179800 + A549 whole cell lysate.

Lane 3 (-): Rabbit monoclonal IgG (ab172730) instead of ab179800 in A549 whole cell lysate.

For western blotting, HRP-conjugated anti-rabbit IgG, specific for the reduced form of IgG, was used as the secondary antibody (1/1500).

Blocking buffer and concentration: 5% NFDM/TBST.

Diluting buffer and concentration: 5% NFDM /TBST.

Western blot analysis on immunoprecipitation pellet from A549 cell lysate using unpurified ab179800.