Anti-p53 antibody [SP5]

• ICC/IF

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Immunocytochemistry/ Immunofluorescence - Anti-p53 antibody [SP5] (AB16665)

Immunocytochemistry/ Immunofluorescence analysis of MCF7 (human breast adenocarcinoma epithelial cell) cells labeling p53 with purified ab16665 at 1/25 (6.5 μg/ml). Cells were fixed in 4% paraformaldehyde and permeabilized with 0.1% Triton X-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 was used as nuclear counterstain. PBS instead of the primary antibody was used as the secondary antibody only control.

This image was generated from the hybridoma version of the product

• Flow Cyt (Intra)

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Flow Cytometry (Intracellular) - Anti-p53 antibody [SP5] (AB16665)

This image was generated from the hybridoma version of the product

Flow cytometry analysis of MCF7 (human breast adenocarcinoma epithelial cell) labeling p53 with purified ab16665 at 1/200 dilution (0.81 μg/ml) (red). Cells were fixed with 4% paraformaldehyde and permeabilised with 90% methanol. Goat anti rabbit IgG (Alexa Fluor^® 488, ab150077) at 1/2000 dilution was used as a secondary antibody. Isotype control - Rabbit monoclonal IgG (ab172730)(black). Unlableled control - Unlabelled cells (blue).

• IHC-P

Lab

Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-p53 antibody [SP5] (AB16665)

Formalin-fixed, paraffin-embedded human colon carcinoma tissue stained for p53 using ab16665 at 1/100 dilution in immunohistochemical analysis.

• Flow Cyt (Intra)

Unknown

Flow Cytometry (Intracellular) - Anti-p53 antibody [SP5] (AB16665)

Flow cytometry overlay histogram showing wild-type HAP1 (green line) and TP53 knockout HAP1 cells stained with ab16665 (red line). The cells were fixed with 4% formaldehyde (10 min) and then permeabilized with 0.1%PBS-Triton X-100 for 15 min. The cells were then incubated in 1x PBS containing 10% normal goat serum to block non-specific protein-protein interaction followed by the antibody (ab16665) (1x10⁶ in 100 μl at 0.008 μg/ml) for 30 min at 22°C.

The secondary antibody Goat anti-rabbit IgG H&L (Alexa Fluor^® 488, pre-adsorbed) (ab150081) was used at 1/2000 for 30 min at 22°C.

Isotype control antibody was Rabbit IgG (monoclonal) (ab172730) used at the same concentration and conditions as the primary antibody (wild-type HAP1 - black line TP53 knockout HAP1 - grey line). Unlabelled sample was also used as a control (this line is not shown for the purpose of simplicity).

Acquisition of >5000 events were collected using a 50 mW Blue laser (488nm) and 525/40 bandpass filter.

This antibody gave a positive signal in TP53 HAP1 knockout cells fixed with 80% methanol (5 min) / permeabilized with 0.1%PBS-Triton X-100 for 15 min used under the same conditions.

• ICC/IF

Lab

Immunocytochemistry/ Immunofluorescence - Anti-p53 antibody [SP5] (AB16665)

ab16665 staining p53 in wild-type Hap1 cells (top panel) and p53 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 ab16665 at 1/25 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™).

This image was generated from the hybridoma version of the product