Recombinant Anti-COX2 / Cyclooxygenase 2 antibody [EPR18377-106] - N-terminal (ab188183)

All lanes : Anti-COX2 / Cyclooxygenase 2 antibody [EPR18377-106] - N-terminal (ab188183) at 1/1000 dilution

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

Lysates/proteins at 20 µg per lane.

Performed under reducing conditions.

Predicted band size: 69 kDa
Observed band size: 74-76 kDa why is the actual band size different from the predicted?

False colour image of Western blot: Anti-COX2 / Cyclooxygenase 2 antibody [EPR18377-106] - N-terminal 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, ab188183 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 : Anti-COX2 / Cyclooxygenase 2 antibody [EPR18377-106] - N-terminal (ab188183) 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.

All lanes : Anti-COX2 / Cyclooxygenase 2 antibody [EPR18377-106] - N-terminal (ab188183) at 1/1000 dilution

Lane 1 : A549 (human lung carcinoma cell line) whole cell lysate
Lane 2 : NIH/3T3 (mouse embryo fibroblast cell line) 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 : Lane 1: 8 seconds; Lane 2: 2 seconds.

Blocking/Dilution buffer: 5% NFDM/TBST.

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 ab188183 at 1/250 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 lipopolysaccharide (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.

Intracellular 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) anduntreated control (green), labeling COX2 / Cyclooxygenase 2 with ab188183 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 ab188183 at 1/30 dilution. Western blot was performed from the immunoprecipitate using ab188183 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: ab188183 IP in NIH/3T3 whole cell lysate. 

Lane 3: Rabbit monoclonal IgG (ab172730) instead of ab188183 in NIH/3T3 whole cell lysate.

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

Exposure: 3 minutes.