Anti-p53 antibody [SP161]

• Flow Cyt (Intra)

Lab

Flow Cytometry (Intracellular) - Anti-p53 antibody [SP161] (AB227655)

Flow cytometry overlay histogram showing wild-type HAP1 (green line) and TP53 knockout HAP1 cells stained with ab227655 (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 (ab227655) (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.

• IHC-P

Lab

Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-p53 antibody [SP161] (AB227655)

ab227655 was shown to react with TP53 in wild-type HAP1 cells in immunohistochemistry with loss of signal observed in a TP53 knockout cell line. HAP1 WT and TP53 KO cells were fixed and paraffin embedded. Sections were mounted onto slides and stained with ab227655 and the corresponding multimer HRP secondary. Chromogen image acquisition was performed, and representative images of each section are shown. Scale bar = 50 µm.

This data was provided by YCharOS Inc., an open science company with the mission of characterizing commercially available antibody reagents for all human proteins. Abcam and YCharOS are working together to help address the reproducibility crisis by enabling the life science community to better evaluate commercially available antibodies.

• IHC-P

Supplier Data

Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-p53 antibody [SP161] (AB227655)

Formalin-fixed paraffin-embedded human bladder transitional cell carcinoma tissue stained for p53 using ab227655 at 1/100 dilution in immunohistochemical analysis.

• ICC/IF

Unknown

Immunocytochemistry/ Immunofluorescence - Anti-p53 antibody [SP161] (AB227655)

Immunocytochemistry/ Immunofluorescence analysis of MCF7 (human breast adenocarcinoma epithelial cell) cells labeling p53 with purified ab227655 at 1/20 (7.25μg/ml). Cells were fixed in 100% Methanol and permeabilized with None. 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.

• IHC-P

Unknown

Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-p53 antibody [SP161] (AB227655)

Formalin-fixed, paraffin-embedded human colon adenocarcinoma tissue stained for p53 using ab227655 at 1/100 dilution in immunohistochemical analysis.

• Flow Cyt (Intra)

Unknown

Flow Cytometry (Intracellular) - Anti-p53 antibody [SP161] (AB227655)

Flow Cytometry analysis of MCF7 (human breast adenocarcinoma epithelial cell) cells labeling p53 with purified ab227655 at 1/20 dilution (7.5 μg/ml) (red). Cells were fixed with 4% paraformaldehyde and permeabilised with 90% methanol. A Goat anti rabbit IgG (Alexa Fluor^® 488, ab150077) secondary antibody was used at 1/2000 dilution. Isotype control - Rabbit monoclonal IgG (ab172730) / Black. Unlabeled control - Unlabelled cells / blue.

• IHC-P

Unknown

Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-p53 antibody [SP161] (AB227655)

Formalin-fixed, paraffin-embedded human stomach adenocarcinoma tissue stained for p53 using ab227655 at 1/100 dilution in immunohistochemical analysis.

• IHC-P

Supplier Data

Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-p53 antibody [SP161] (AB227655)

Formalin-fixed paraffin-embedded human breast ductal carcinoma tissue stained for p53 using ab227655 at 1/100 dilution in immunohistochemical analysis.

• IHC-P

Supplier Data

Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-p53 antibody [SP161] (AB227655)

Formalin-fixed, paraffin-embedded human B-cell lymphoma tissue stained for p53 using ab227655 at 1/100 dilution in immunohistochemical analysis.

• IHC-P

Supplier Data

Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-p53 antibody [SP161] (AB227655)

Formalin-fixed paraffin-embedded human ovarian adenocarcinoma tissue stained for p53 using ab227655 at 1/100 dilution in immunohistochemical analysis.

• IHC-P

Supplier Data

Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-p53 antibody [SP161] (AB227655)

Formalin-fixed paraffin-embedded human lung squamous cell carcinoma tissue stained for p53 using ab227655 at 1/100 dilution in immunohistochemical analysis.

• WB

Supplier Data

Western blot - Anti-p53 antibody [SP161] (AB227655)

ab227655 was shown to react with TP53 in wild-type HAP1 cells in Western blot with loss of signal observed in a TP53 knockout cell line. Wild-type HAP1 and TP53 knockout cell lysates were subjected to SDS-PAGE. Membranes were blocked in 5% milk in TBST for 1 hr before incubation with ab227655 overnight at 4 °C at a 1/500 dilution. Blots were incubated with secondary antibodies at 0.2 µg/mL before imaging.

This data was provided by YCharOS Inc., an open science company with the mission of characterizing commercially available antibody reagents for all human proteins. Abcam and YCharOS are working together to help address the reproducibility crisis by enabling the life science community to better evaluate commercially available antibodies.

All lanes:

Western blot - Anti-p53 antibody [SP161] (ab227655) at 1/500 dilution

Lane 1:

Wild-type HAP1 lysate at 20 µg at 20 µg

Lane 2:

TP53 knock-out HAP1 lysate at 20 µg

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