ab33333 staining Cortactin in wild-type HAP1 cells (top panel) and CTTN knockout HAP1 cells (bottom panel). The cells were fixed with 100% methanol (5min), 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 ab33333 at 1μg/ml and ab7291 at 1μg/ml 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 goat secondary antibody to Mouse IgG (Alexa Fluor® 594) (ab150120) at 2 μg/ml (shown in pseudo colour red). Nuclear DNA was labelled in blue with DAPI.

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

All lanes :

Lane 1 : HAP1 whole cell lysate
Lane 2 : HAP1 CTTN whole cell lysate
Lane 3 : NIH3T3 whole cell lysate
Lane 4 : A431 whole cell lysate
Lane 5 : PC12 whole cell lysate

Lysates/proteins at 20 µg per lane.

Performed under reducing conditions.

Predicted band size: 61 kDa
Observed band size: 80 kDa why is the actual band size different from the predicted?

ab33333 was shown to specifically react with CTTN (Cortactin) in wild type HAP1 cells. No band was observed when CTTN (Cortactin) knockout samples were used. Wild-type and CTTN (Cortactin) knockout samples were subjected to SDS-PAGE. ab33333 and ab181602 (Rabbit anti GAPDH) were incubated overnight at 4°C at a 1ug/ml concentration and 1/10000 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 1/20000 dilution for 1 hour at room temperature before imaging.

Anti-Cortactin antibody [4F11] (ab33333) at 1/1000 dilution + HEK293T cells at 10 µg

Secondary
Polyclonal goat anti-mouse HRP at 1/10000 dilution

Predicted band size: 61 kDa

Samples were incubated with primary antibody for 2 hours at 20ºC.

Blocking: 5% milk for 1 hour at 20ºC.

See Abreview

Anti-Cortactin antibody [4F11] (ab33333) at 1/1000 dilution + Bovine aortic endothelial whole cell lysates at 10 µg

Secondary
Goat anti-mouse polyclonal HRP conjugate at 1/10000 dilution

Developed using the ECL technique.

Performed under reducing conditions.

Predicted band size: 61 kDa
Additional bands at: 80 kDa (possible post-translational modification)


Exposure time: 5 seconds

Blocking was with 5% milk for 1 hour at 20°C.

See Abreview

ab33333 staining Cortactin (red) in Human palladin-activated fibroblasts by Immunocytochemistry/ Immunofluorescence.

Cells were fixed with paraformaldehyde and permeabilized with 0.2% Triton X-100. Samples were incubated with primary antibody (1/500 in diluent) before incubation with a Rhodamine Red IgG (1/100).

IHC image of Cortactin staining in a section of formalin-fixed paraffin-embedded normal human breast carcinoma* performed on a Leica BOND^TM system using the standard protocol F. The section was pre-treated using heat mediated antigen retrieval with sodium citrate buffer (pH6, epitope retrieval solution 1) for 20mins. The section was then incubated with ab33333, 0.05ug/ml, for 15 mins at room temperature and detected using an HRP conjugated compact polymer system. DAB was used as the chromogen. The section was then counterstained with haematoxylin and mounted with DPX. The inset secondary-only control image is taken from an identical assay without primary antibody.

For other IHC staining systems (automated and non-automated) customers should optimize variable parameters such as antigen retrieval conditions, primary antibody concentration and antibody incubation times.