All lanes : Anti-Cortactin antibody [EP1922Y] (ab81208) at 1/50000 dilution

Lane 1 : Wild-type HEK-293T cell lysate
Lane 2 : CTNN knockout HEK-293T cell lysate

Lysates/proteins at 20 µg per lane.

Performed under reducing conditions.

Predicted band size: 62 kDa
Observed band size: 70 kDa why is the actual band size different from the predicted?

Lanes 1-2: Merged signal (red and green). Green - ab81208 observed at 70 kDa. Red - loading control ab8245 observed at 37 kDa.

 ab81208 Anti-Cortactin antibody [EP1922Y] was shown to specifically react with Cortactin in wild-type HEK-293T cells. Loss of signal was observed when knockout cell line ab266819 (knockout cell lysate ab257147) was used. Wild-type and Cortactin knockout samples were subjected to SDS-PAGE. ab81208 and Anti-GAPDH antibody [6C5] - Loading Control (ab8245) were incubated overnight at 4°C at 1 in 50000 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.

 

ab81208 staining Cortactin in wild-type HAP1 cells (top panel) and Cortactin knockout HAP1 cells (bottom panel). The cells were fixed with 4% formaldehyde (10min), 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 ab81208 at 1/1000 dilution and ab195889 at 1/250 dilution (shown in pseudo colour red) 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). Nuclear DNA was labelled in blue with DAPI.

Image was taken with a confocal microscope (Leica-Microsystems, TCS SP8).

All lanes : Anti-Cortactin antibody [EP1922Y] (ab81208) at 1/2000 dilution

Lane 1 : Wild-type HAP1 cell lysate
Lane 2 : Cortactin knockout HAP1 cell lysate
Lane 3 : NIH3T3 cell lysate
Lane 4 : A431 cell lysate

Lysates/proteins at 20 µg per lane.

Predicted band size: 62 kDa

Lanes 1 - 4: Merged signal (red and green). Green - ab81208 observed at 62 kDa. Red - loading control, ab8245, observed at 37 kDa.

ab81208 was shown to specifically react with Cortactin in wild-type HAP1 cells along with additional cross reactive bands. No band was observed when Cortactin knockout samples were examined. Wild-type and Cortactin knockout samples were subjected to SDS-PAGE. ab81208 and ab8245 (loading control to GAPDH) were both diluted 1/2000 and incubated overnight at 4°C. 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 1/10,000 dilution for 1 hour at room temperature before imaging.

Purified ab81208 at 1/50 dilution (2µg) immunoprecipitating Cortactin in HeLa whole cell lysate.
Lane 1 (input): HeLa (Human cervix adenocarcinoma epithelial cell) whole cell lysate 10µg
Lane 2 (+): ab81208 + HeLa whole cell lysate.
Lane 3 (-): Rabbit monoclonal IgG (ab172730) instead of ab81208 in HeLa 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: 62 kDa

Anti-Cortactin antibody [EP1922Y] (ab81208) at 1/100000 dilution + HeLa cell lysate at 10 µg

Secondary
HRP labelled Goat anti-Rabbit at 1/2000 dilution

Predicted band size: 62 kDa

Immunohistochemical analysis of paraffin-embedded human breast carcinoma staining Cortacin with ab81208 at 1/100 dilution. Heat mediated antigen retrieval with Tris-EDTA (pH 9) was perfomed.

Heat mediated antigen retrieval was performed with citrate buffer pH 6 before commencing with IHC staining protocol.

Immunofluorescent staining of MCF7 cells using 1/100 ab81208

Overlay histogram showing HeLa cells stained with ab81208 (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 (ab81208, 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.