Anti-DAB2 antibody [EP2297Y]

• ICC/IF

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Immunocytochemistry/ Immunofluorescence - Anti-DAB2 antibody [EP2297Y] (AB76253)

ab76253 (unpurified) stained HeLa cells. The cells were 4% formaldehyde fixed for 10 minutes at room temperature and then incubated in 1%BSA / 10% normal goat serum / 0.3M glycine in 0.1% PBS-Tween for 1hour at room temperature to permeabilise the cells and block non-specific protein-protein interactions. The cells were then incubated with the antibody (unpurified ab76253 at 1/100 dilution) overnight at +4°C. The secondary antibody (pseudo-colored green) was Goat Anti-Rabbit IgG H&L (Alexa Fluor® 488) preadsorbed (ab150081) used at a 1/1000 dilution for 1hour at room temperature. Alexa Fluor® 594 WGA was used to label plasma membranes (pseudo-colored red) at a 1/200 dilution for 1hour at room temperature. DAPI was used to stain the cell nuclei (pseudo-colored blue) at a concentration of 1.43μM for 1hour at room temperature.

• Flow Cyt (Intra)

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Flow Cytometry (Intracellular) - Anti-DAB2 antibody [EP2297Y] (AB76253)

Overlay histogram showing HeLa cells stained with unpurified ab76253 (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 (ab76253 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 (monclonal) (1μg/1x10⁶ cells) used under the same conditions. Acquisition of >5000 events was performed. This antibody gave a positive signal in HeLa cells fixed with 4% paraformaldehyde (10 min)/permeabilized with 0.1% PBS-Tween for 20 min used under the same conditions.

• ICC/IF

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Immunocytochemistry/ Immunofluorescence - Anti-DAB2 antibody [EP2297Y] (AB76253)

Immunocytochemistry/ Immunofluorescence analysis of HeLa (Human cervix adenocarcinoma epithelial cell) cells labeling DAB2 with purified ab76253 at 1/100 dilution (1.1 μg/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.

• Flow Cyt (Intra)

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Flow Cytometry (Intracellular) - Anti-DAB2 antibody [EP2297Y] (AB76253)

Intracellular Flow Cytometry analysis of HeLa (Human cervix adenocarcinoma epithelial cell) cells labeling DAB2 with purified ab76253 at 1/100 dilution (1μg/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).

• IP

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Immunoprecipitation - Anti-DAB2 antibody [EP2297Y] (AB76253)

Purified ab76253 at 1/20 dilution (0.5 μg) immunoprecipitating DAB2 in HeLa whole cell lysate.

Lane 1 (input) : HeLa (Human cervix adenocarcinoma epithelial cell) whole cell lysate 10 μg

Lane 2 (+) : ab76253 + HeLa whole cell lysate.

Lane 3 (-) : Rabbit monoclonal IgG (ab172730) instead of ab76253 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 : 105 kDa

All lanes:

Immunoprecipitation - Anti-DAB2 antibody [EP2297Y] (ab76253)

Predicted band size: 82 kDa

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• WB

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Western blot - Anti-DAB2 antibody [EP2297Y] (AB76253)

All lanes:

Western blot - Anti-DAB2 antibody [EP2297Y] (ab76253) at 1/1000 dilution

Lane 1:

PC-3 (Human prostate adenocarcinoma epithelial cell) whole cell lysate at 20 µg

Lane 2:

HeLa (Human cervix adenocarcinoma epithelial cell) whole cell lysate at 20 µg

Secondary

All lanes:

Goat Anti-Rabbit IgG (HRP) with minimal cross-reactivity with human IgG at 1/2000 dilution

Predicted band size: 82 kDa

Observed band size: 105 kDa

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