Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) analysis of rat kidney tissue sections labeling COX1 / Cyclooxygenase 1 with Purified ab109025 at 1:150 dilution. Heat mediated antigen retrieval was performed using ab93684 (Tris/EDTA buffer, pH 9.0). Tissue was counterstained with Hematoxylin. ImmunoHistoProbe one step HRP Polymer (ready to use) secondary antibody was used at 1:0 dilution.

PBS instead of the primary antibody was used as the negative control.

All lanes : Anti-COX1 / Cyclooxygenase 1 antibody [EPR5866] (ab109025) at 1/10000 dilution (purified)

Lane 1 : A431 (Human epidermoid carcinoma epithelial cell) whole cell lysates
Lane 2 : L6 (Rat skeletal muscle myoblast) whole cell lysates

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

Blocking and diluting buffer: 5% NFDM/TBST.

All lanes : Anti-COX1 / Cyclooxygenase 1 antibody [EPR5866] (ab109025) at 1/2000 dilution (purified)

Lane 1 : HaCaT (Human skin keratinocyte) whole cell lysates
Lane 2 : NIH/3T3 (Mouse embryonic fibroblast) whole cell lysates

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

Blocking and diluting buffer: 5% NFDM/TBST.

ab109025 (purified) at 1:20 dilution (0.8μg) immunoprecipitating COX1 / Cyclooxygenase 1 in C2C12 whole cell lysate.

Lane 1 (input): C2C12 (Mouse myoblasts myoblast) whole cell lysate,10μg
Lane 2 (+): ab109025 & C2C12 whole cell lysate
Lane 3 (-): Rabbit monoclonal IgG (ab172730) instead of ab109025 in C2C12 whole cell lysate

For western blotting, VeriBlot for IP Detection Reagent (HRP) (ab131366) was used for detection at 1:1000 dilution.

Blocking and diluting buffer: 5% NFDM/TBST.

Flow Cytometry analysis of NIH/3T3 (Mouse embryonic fibroblast) cells labeling COX1 / Cyclooxygenase 1 with purified ab109025 at 1:100 dilution (red). Cells were fixed with 4% Paraformaldehyde. A Goat anti rabbit IgG (Alexa Fluor^® 488) secondary antibody was used at 1:2000 dilution. Isotype control - Rabbit monoclonal IgG (Black). Unlabeled control - Cell without incubation with primary antibody and secondary antibody (Blue).

Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) analysis of mouse kidney tissue sections labeling COX1 / Cyclooxygenase 1 with Purified ab109025 at 1:150 dilution. Heat mediated antigen retrieval was performed using ab93684 (Tris/EDTA buffer, pH 9.0). Tissue was counterstained with Hematoxylin. ImmunoHistoProbe one step HRP Polymer (ready to use) secondary antibody was used at 1:0 dilution.

PBS instead of the primary antibody was used as the negative control.

Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) analysis of human cerebrum tissue sections labeling COX1 / Cyclooxygenase 1 with Purified ab109025 at 1:150 dilution. Heat mediated antigen retrieval was performed using ab93684 (Tris/EDTA buffer, pH 9.0). Tissue was counterstained with Hematoxylin. ImmunoHistoProbe one step HRP Polymer (ready to use) secondary antibody was used at 1:0 dilution.

PBS instead of the primary antibody was used as the negative control.

Immunocytochemistry/Immunofluorescence analysis of Neuro-2a (mouse neuroblastoma) labelling COX1 with purified ab109025 at 1/50. Cells were fixed with 100% methanol. An Alexa Fluor^® 488-conjugated goat anti-rabbit IgG (1/1000) was used as the secondary antibody (ab150077). Nuclei counterstained with DAPI (blue).

Control: PBS only.

All lanes : Anti-COX1 / Cyclooxygenase 1 antibody [EPR5866] (ab109025) at 1/1000 dilution (unpurified)

Lane 1 : NIH/3T3 cell lysate
Lane 2 : HaCaT cell lysate
Lane 3 : Neuro 2a cell lysate
Lane 4 : C2C12 cell lysate
Lane 5 : A431 cell lysate
Lane 6 : L6 cell lysate

Lysates/proteins at 10 µg per lane.

Predicted band size: 69 kDa

Unpurified ab109025 at 1/250 dilution staining COX1 / Cyclooxygenase 1 in human skin by immunohistochemistry, paraffin-embedded tissue.

Unpurified ab109025 at 1/100 dilution staining COX1 / Cyclooxygenase 1 in HeLa cells by Immunofluorescence.

Overlay histogram showing NIH/3T3 cells stained with unpurified ab109025 (red line). The cells were fixed with 80% methanol (5 min) and then permeabilized with 0.1% PBS-Tween for 20 min. The cells were then incubated in 1x PBS / 10% normal goat serum / 0.3M glycine to block non-specific protein-protein interactions followed by the antibody (ab109025, 1/100 dilution) for 30 min at 22ºC. The secondary antibody used was DyLight^® 488 goat anti-rabbit IgG (H+L) (ab96899) at 1/500 dilution for 30 min at 22ºC. Isotype control antibody (black line) was rabbit IgG (monoclonal) (1µg/1x10⁶ cells) used under the same conditions. Acquisition of >5,000 events was performed.

Equilibrium disassociation constant (K_D)
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