All lanes : Anti-COX2 / Cyclooxygenase 2 antibody [EPR12012] (ab179800) at 1/1000 dilution

Lane 1 : A549 cell lysate
Lane 2 : U-87 MG cell lysate
Lane 3 : Wild-type HeLa cell lysate
Lane 4 : PTGS2 knockout HeLa cell lysate

Lysates/proteins at 20 µg per lane.

Predicted band size: 69 kDa

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.

 

Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) analysis of mouse kidney tissue sections labeling COX2 / Cyclooxygenase 2 with purified ab179800 at 1/4000 dilution (0.125 µg/ml).
Heat mediated antigen retrieval was performed with Tris-EDTA buffer (pH 9.0, epitope retrieval solution2) for 20 mins. Rabbit specific IHC polymer detection kit HRP/DAB (ab209101) was used as the secondary antibody. Negative control: secondary antibody only control. Hematoxylin was used as a counterstain.

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）

Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) analysis of mouse liver tissue sections labeling COX2 / Cyclooxygenase 2 with purified ab179800 at 1/4000 dilution (0.125 µg/ml).
Heat mediated antigen retrieval was performed with Tris-EDTA buffer (pH 9.0, epitope retrieval solution2) for 20 mins. Rabbit specific IHC polymer detection kit HRP/DAB (ab209101) was used as the secondary antibody. Negative control: secondary antibody only control. Hematoxylin was used as a counterstain.

All lanes : Anti-COX2 / Cyclooxygenase 2 antibody [EPR12012] (ab179800) at 1/1000 dilution (Purified)

Lane 1 : U-87 MG (human glioblastoma-astrocytoma epithelial cell line) whole cell lysate with 5% NFDM/TBST
Lane 2 : HCT 116 (human colorectal carcinoma cell line) whole cell lysate with 5% NFDM/TBST
Lane 3 : MCF7 (human breast adenocarcinoma cell line) whole cell lysate with 5% NFDM/TBST

Lysates/proteins at 20 µg per lane.

Secondary
All lanes : Goat Anti-Rabbit IgG H&L (HRP) (ab97051) at 1/20000 dilution

Predicted band size: 69 kDa
Observed band size: 72 kDa why is the actual band size different from the predicted?

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.

Anti-COX2 / Cyclooxygenase 2 antibody [EPR12012] (ab179800) at 1/5000 dilution (purified) + Mouse spleen tissue lysate at 20 µg

Secondary
HRP-conjugated anti-rabbit IgG, specific to the non-reduced form of IgG at 1/50000 dilution

Predicted band size: 69 kDa
Observed band size: 72 kDa why is the actual band size different from the predicted?

Blocking and dilution buffer: 5% NFDM/TBST.

Anti-COX2 / Cyclooxygenase 2 antibody [EPR12012] (ab179800) at 1/5000 dilution (purified) + A549 whole cell lysate at 20 µg

Secondary
HRP-conjugated anti-rabbit IgG, specific to the non-reduced form of IgG at 1/50000 dilution

Predicted band size: 69 kDa
Observed band size: 72 kDa why is the actual band size different from the predicted?

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.

Anti-COX2 / Cyclooxygenase 2 antibody [EPR12012] (ab179800) at 1/1000 dilution (unpurified) + A549 cell lysate at 10 µg

Predicted band size: 69 kDa

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