Anti-p53 antibody [SP5]

• 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

Supplier Data

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

ab16665 was shown to react with TP53 in wild-type HAP1 cells in immunocytochemistry with loss of signal observed in a TP53 knockout cell line. Wild-type and knockout cells were mixed and pelleted at a 1 : 1 ratio on coverslips. The cells were fixed with 4% paraformaldehyde (15 min) then permeabilized with 0.1% Triton X-100 (10min) and then blocked with 1x PBS, 0.01% Triton X-100, 5% BSA, 5% NGS. The cells were then incubated with ab16665 at 1/100 dilution overnight at 4°C followed by a further incubation at room temperature for 1h with a goat anti-rabbit secondary antibody to (Alexa Fluor® 555) at 0.5 μg/ml. Acquisition of the green (wild-type), red (antibody staining) and far-red (knockout) channels was performed. Representative grayscale images of the red channel are shown. Wild-type and knockout cells are outlined with yellow and magenta dashed line, respectively. Schematic representation of the mosaic strategy used is shown on the bottom-right panel. Image was acquired with a Zeiss(LSM-880).

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.

• Flow Cyt (Intra)

Unknown

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.

• 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

• ICC/IF

Unknown

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

• IP

Supplier Data

Immunoprecipitation - Anti-p53 antibody [SP5] (AB16665)

Immunoprecipitation of TP53 in HAP1 cells. Lysates were prepared and immunoprecipitation was performed using 2 μg of ab16665 pre-coupled to Protein A beads. Samples were then washed and processed for western blot.

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.

• WB

Supplier Data

Western blot - Anti-p53 antibody [SP5] (AB16665)

ab16665 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 ab16665 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 [SP5] (ab16665) at 1/500 dilution

Lane 1:

Wild-type HAP1 lysate at 20 µg

Lane 2:

TP53 knock-out HAP1 lysate at 20 µg

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