Recombinant Anti-c-Jun antibody [EP693Y] (ab40766)

Purified ab40766 at 1/20 dilution (1µg) immunoprecipitating c-Jun in NIH/3T3 whole cell lysate.
Lane 1 (input): NIH/3T3 (Mouse embryonic fibroblast) whole cell lysate 10µg
Lane 2 (+): ab40766 + NIH/3T3 whole cell lysate.
Lane 3 (-): Rabbit monoclonal IgG (ab172730) instead of ab40766 in NIH/3T3 whole cell lysate.
VeriBlot for IP Detection Reagent (HRP) (ab131366) (1/1000 dilution) was used for Western blotting.
Blocking Buffer and concentration: 5% NFDM/TBST.
Diluting buffer and concentration: 5% NFDM/TBST.
Observed band size: 39 kDa

Blocking Buffer and concentration: 5% NFDM/TBST

Immunocytochemistry analysis of NIH/3T3 (Mouse embryonic fibroblast) cells labeling c-Jun with Purified ab40766 at 1:50 dilution (4.06 ?g/ml). Cells were fixed in 4% Paraformaldehyde and permeabilized with 0.1% tritonX-100. Cells were counterstained with Ab195889 Anti-alpha Tubulin antibody [DM1A] - Microtubule Marker (Alexa Fluor^® 594) 1:200 (2.5 µg/ml). Goat anti rabbit IgG (Alexa Fluor^® 488, ab150077) was used as the secondary antibody at 1:1000 (2 µg/ml) dilution. DAPI (blue) was used as nuclear counterstain. PBS instead of the primary antibody was used as the secondary antibody only control.

Intracellular Flow Cytometry analysis of HEK-293 (Human embryonic kidney epithelial cell) cells labeling c-Jun with Purified ab40766 at 1/20 dilution (10µg/ml) (Red). Cells were fixed with 4% Paraformaldehyde and permeabilised with 90% Methanol. A Goat anti rabbit IgG (Alexa Fluorr^® 488, ab150077) secondary antibody was used at 1/2000. Isotype control - Rabbit monoclonal IgG (Black). Unlabeled control - Cell without incubation with primary antibody and secondary antibody (Blue). 

ab40766 staining c-Jun in wild-type HEK293 cells (top panel) and c-Jun knockout HEK293 cells (bottom panel). The cells were fixed with 100% methanol (5 min), permeabilized with 0.1% Triton X-100 for 5 minutes and then blocked with 1% BSA/10% normal goat serum/0.3M glycine in 0.1% PBS-Tween for 1h. The cells were then incubated with ab40766 at 1/250 dilution and ab7291 (Mouse monoclonal to alpha Tubulin) at 1/1000 dilution overnight at +4°C, followed by a further incubation at room temperature for 1h with a goat secondary antibody to rabbit IgG (Alexa Fluor® 488) (ab150081) at 2 μg/ml (shown in green) and a goat secondary antibody to mouse IgG (Alexa Fluor® 594) (ab150120) at 2 μg/ml (shown in pseudo color red). Nuclear DNA was labelled in blue with DAPI.

Image was taken with a high-content analysis system (Perkin Elmer, Operetta CLS™).

Lanes 1 - 2: Merged signal (red and green). Green - ab40766 observed at 35 kDa. Red - loading control, ab18058, observed at 130 kDa.

ab40766 was shown to specifically react with Jun in wild-type HEK-293 cells as signal was lost in Jun knockout cells. Wild-type and Jun knockout samples were subjected to SDS-PAGE. The membrane was blocked with 3% Milk. Ab40766 and ab18058 (Mouse anti-Vinculin loading control) were incubated overnight at 4°C at 1/1000 dilution and 1/10000 dilution respectively. Blots were developed with Goat anti-Rabbit IgG H&L (IRDye^® 800CW) preabsorbed ab216773 and Goat anti-Mouse IgG H&L (IRDye^® 680RD) preabsorbed ab216776 secondary antibodies at 1/10000 dilution for 1 hour at room temperature before imaging.