Anti-Cortactin antibody [EP1922Y]

• IHC-P

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Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-Cortactin antibody [EP1922Y] (AB81208)

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.

• Flow Cyt (Intra)

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Flow Cytometry (Intracellular) - Anti-Cortactin antibody [EP1922Y] (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 22°C. The secondary antibody used was DyLight® 488 goat anti-rabbit IgG (H+L) (ab96899) at 1/500 dilution for 30 min at 22°C. Isotype control antibody (black line) was rabbit IgG (monoclonal) (1μg/1x10⁶ cells) used under the same conditions. Acquisition of >5,000 events was performed.

• ICC/IF

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Immunocytochemistry/ Immunofluorescence - Anti-Cortactin antibody [EP1922Y] (AB81208)

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).

• ICC/IF

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Immunocytochemistry/ Immunofluorescence - Anti-Cortactin antibody [EP1922Y] (AB81208)

Immunofluorescent staining of MCF7 cells using 1/100 ab81208

• IP

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Immunoprecipitation - Anti-Cortactin antibody [EP1922Y] (AB81208)

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

All lanes:

Immunoprecipitation - Anti-Cortactin antibody [EP1922Y] (ab81208)

Predicted band size: 62 kDa

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• WB

Lab

Western blot - Anti-Cortactin antibody [EP1922Y] (AB81208)

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.

All lanes:

Western blot - Anti-Cortactin antibody [EP1922Y] (ab81208) at 1/50000 dilution

Lane 1:

Wild-type HEK-293T cell lysate at 20 µg

Lane 2:

CTNN knockout HEK-293T cell lysate at 20 µg

Lane 2:

Western blot - Human CTTN (Cortactin) knockout HEK-293T cell line (<a href='/en-us/products/cell-lines/human-cttn-cortactin-knockout-hek-293t-cell-line-ab266819'>ab266819</a>)

Predicted band size: 62 kDa

Observed band size: 70 kDa

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• WB

Lab

Western blot - Anti-Cortactin antibody [EP1922Y] (AB81208)

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.

All lanes:

Western blot - Anti-Cortactin antibody [EP1922Y] (ab81208) at 1/2000 dilution

Lane 1:

Wild-type HAP1 cell lysate at 20 µg

Lane 2:

Cortactin knockout HAP1 cell lysate at 20 µg

Lane 3:

NIH3T3 cell lysate at 20 µg

Lane 4:

A431 cell lysate at 20 µg

Predicted band size: 62 kDa

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• WB

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Western blot - Anti-Cortactin antibody [EP1922Y] (AB81208)

All lanes:

Western blot - Anti-Cortactin antibody [EP1922Y] (ab81208) at 1/100000 dilution

All lanes:

HeLa cell lysate at 10 µg

Secondary

All lanes:

HRP labelled Goat anti-Rabbit at 1/2000 dilution

Predicted band size: 62 kDa

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• WB

CiteAb

Western blot - Anti-Cortactin antibody [EP1922Y] (AB81208)

Cortactin western blot using anti-Cortactin antibody [EP1922Y] ab81208. Publication image and figure legend from Li, Y., Fu, Y., et al., 2019, Cell Death Dis, PubMed 31138777.

ab81208 was used in this publication in western blot. This may not be the same as the application(s) guaranteed by Abcam. For a full list of applications guaranteed by Abcam for ab81208 please see the product overview.

Effects of the HBx-CTTN interaction on cell proliferation and migration.a HepG2 and MHCC-LM3 cells were co-transfected with si-CTTN and pcDNA3.1( + )-HBx-overexpressing vectors. Protein expressions of HBx and CTTN were measured by western blot. b, c HepG2 and MHCC-LM3 cells were co-transfected with si-CTTN and pcDNA3.1( + )-HBx-overexpressing vectors and the cell viability was examined using MTT assays. d, e DNA synthesis was examined in BrdU assays; the data are presented as the mean ± SD of three independent experiments. f, g Migration of the indicated groups of cells examined using transwell assays on a light microscope. The statistical analysis was shown in the right panel. *p < 0.05, **p < 0.01

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