Anti-COX2 / Cyclooxygenase 2 antibody [EPR18376-119] - C-terminal (ab188184) at 1/1000 dilution + NIH/3T3 (mouse embryo fibroblast cell line) whole cell lysate at 10 µg

Secondary
Goat Anti-Rabbit IgG H&L (HRP) (ab97051) at 1/100000 dilution

Developed using the ECL technique.

Predicted band size: 69 kDa
Observed band size: 74 kDa why is the actual band size different from the predicted?


Exposure time: 3 seconds

Blocking/Dilution buffer: 5% NFDM/TBST.

All lanes : Anti-COX2 / Cyclooxygenase 2 antibody [EPR18376-119] - C-terminal (ab188184) at 1/1000 dilution

Lane 1 : Untreated RAW 264.7 (mouse macrophage cell line transformed with Abelson murine leukemia virus) whole cell lysate
Lane 2 : RAW 264.7 treated with 1 µg/ml LPS for 6 hours whole cell lysate

Lysates/proteins at 20 µg per lane.

Secondary
All lanes : Goat Anti-Rabbit IgG H&L (HRP) (ab97051) at 1/100000 dilution

Developed using the ECL technique.

Predicted band size: 69 kDa
Observed band size: 74 kDa why is the actual band size different from the predicted?


Exposure time: 1 second

Blocking/Dilution buffer: 5% NFDM/TBST.

The expression profile and molecular weight observed is consistent with what has been described in the literature (PMID: 9737714). COX2 protein expression is induced by LPS in neutrophils.

Immunofluorescent analysis of 4% paraformaldehyde-fixed, 0.1% Triton X-100 permeabilized RAW 264.7 (mouse macrophage cell line transformed with Abelson murine leukemia virus) cells, untreated or treated with lipopolysaccharide (1 µg/ml) for 6 hours, labeling COX2 / Cyclooxygenase 2 with ab188184 at 1/150 dilution, followed by Goat Anti-Rabbit IgG H&L (Alexa Fluor^® 488) (ab150077) secondary antibody at 1/1000 dilution (green). Confocal image showing increased cytoplasmic staining on RAW 264.7 cells treated with lipopolysaccharides (1 µg/ml) for 6 hours.

The nuclear counter stain is DAPI (blue). Tubulin is detected with Anti-alpha Tubulin antibody [DM1A] - Microtubule Marker (Alexa Fluor^® 594) (ab195889) (red) at 1/200 dilution.

Secondary antibody only control: Used PBS instead of primary antibody, secondary antibody is Goat Anti-Rabbit IgG H&L (Alexa Fluor^® 488) (ab150077) secondary antibody at 1/1000 dilution.

Flow cytometric analysis of 4% paraformaldehyde-fixed, 90% methanol permeabilized RAW 264.7 (mouse macrophage cell line transformed with Abelson murine leukemia virus) cell line, treated with 1μg/ml LPS for 6h (red)  and untreated control (green), labeling COX2 / Cyclooxygenase 2 with ab188184 at 1/600 dilution compared with a Rabbit IgG, monoclonal [EPR25A] - Isotype Control (ab172730) (black) and an unlabeled control (cells without incubation with primary antibody and secondary antibody) (blue). Goat Anti-Rabbit IgG H&L (Alexa Fluor^® 488) (ab150077) at 1/2000 dilution was used as the secondary antibody.

COX2 / Cyclooxygenase 2 was immunoprecipitated from 0.35 mg of NIH/3T3 (mouse embryo fibroblast cell line) whole cell lysate with ab188184 at 1/30 dilution. Western blot was performed from the immunoprecipitate using ab188184 at 1/1000 dilution. VeriBlot for IP Detection Reagent (HRP) (ab131366), was used for detection at 1/10000 dilution.

Lane 1: NIH/3T3 whole cell lysate 10μg (Input). 

Lane 2: ab188184 IP in NIH/3T3 whole cell lysate. 

Lane 3: Rabbit monoclonal IgG (ab172730) instead of ab188184 in NIH/3T3 whole cell lysate.

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

Exposure: 30 seconds.