Lanes 1, 5 and 9: Wild-type HAP1 cell lysate (20 µg)
Lanes 2, 6 and 10: p53 knockout HAP1 cell lysate (20 µg)
Lanes 3, 7 and 11: A431 cell lysate (20 µg)
Lanes 4, 8 and 12: Saos-2 cell lysate (20 µg)
Lanes 1, 2, 3 and 4: Green signal from target – ab154036 observed at 53 kDa
Lanes 5, 6, 7 and 8: Red signal from loading control – ab181602 observed at 37 kDa
Lanes 9, 10, 11 and 12: Merged (red and green) signal

ab154036 was shown to specifically react with p53 in wild type HAP1 cells. No band was observed when p53 knockout samples were used. Wild-type and p53 knockout samples were subjected to SDS-PAGE. ab154036 and ab181602 (loading control to GAPDH) were diluted 2 µg/mL and 1/10000 respectively and incubated overnight at 4ºC. Blots were developed with Goat anti-Mouse IgG H&L (IRDye^® 800CW) preadsorbed ab216772 and Goat Anti-Rabbit IgG H&L (IRDye^® 680RD) preadsorbed ab216777 secondary antibodies at 1/10000 dilution for 1 h at room temperature before imaging.

ab154036 staining p53 in wild-type HAP1 cells (top panel) and p53 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 ab154036 at 1μg/ml concentration and ab202272 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 Mouse IgG (Alexa Fluor® 488) (ab150117) at 2 μg/ml (shown in green). Nuclear DNA was labelled in blue with DAPI.

IHC image of ab154036 staining beta Catenin in human colon adenocarcinoma formalin-fixed paraffin-embedded tissue sections*, performed on a Leica Bond. The section was pre-treated using heat mediated antigen retrieval with sodium citrate buffer (pH6, epitope retrieval solution 1) for 20 mins. The section was then incubated with ab154036, 1/500 dilution, for 15 mins at room temperature and detected using an HRP conjugated compact polymer system. DAB was used as the chromogen. The section was then counterstained with haematoxylin and mounted with DPX. High magnification of the tumor region - T (lower right panel) and adjacent normal crypts - N (lower left panel) are shown.
For other IHC staining systems (automated and non-automated) customers should optimize variable parameters such as antigen retrieval conditions, primary antibody concentration and antibody incubation times.
*Tissue obtained from the Human Research Tissue Bank, supported by the NIHR Cambridge Biomedical Research Centre

Figure 2: p53 (ab154036) antibody specificity demonstrated by immunoprecipitation, followed by Western blot

Lane 1: Hek293 whole cell extract
Lane 2: anti-p53 (ab154036) IP using Hek293 cells extracted with RIPA buffer
Lane 3: anti-p53 (ab154036) IP using Hek293 cells extracted with Lauryl Maltoside (ab109857)

Western blot with anti-p53 (ab32389) 1/1000.

Secondary goat anti-rabbit IgG 1/5000.

Figure 4: In-cell ELISA with anti-p53 antibody (ab154036)

Standard In-Cell ELISA protocol was performed on vehicle- and camptothecin-treated MCF7 cells using 1 µg/mL anti-p53 primary antibody and IR800-conjugated goat anti-mouse IgG secondary antibody. Cells were imaged using a LI-COR® Odyssey near-infrared scanner. The data is presented as background-subtracted IR800 signals (A) or Janus green-normalized signals (B).

Figure 5: Flow Cytometry with anti-p53 antibody (ab154036) using Hek293 cells
Standard flow cytometry procedure was performed on Hek293 cells that were fixed and permeabilized with methanol and stained with 1 µg/mL of anti-p53 antibody (red) or a negative isotype control antibody (black). 1% BSA in PBS was used as the blocking reagent for all the blocking steps.

All lanes : Anti-p53 antibody [9D3DE3] (ab154036) at 2 µg/ml

Lane 1 : Recombinant Human p53 protein (ab43615) at 0.002 µg
Lane 2 : Hek293 cells at 40 µg
Lane 3 : 6 hour vehicle-treated MCF7 cells at 40 µg
Lane 4 : 6 hour camptothecin-treated MCF7 cells at 40 µg

Secondary
All lanes : Goat polyclonal to Mouse IgG - HRP at 1/5000 dilution

Observed band size: 53 kDa why is the actual band size different from the predicted?

Figure 3: Immunocytochemistry with anti-p53 antibody (ab154036) using camptothecin-treated MCF7 cells

MCF7 cells were treated with vehicle (A, B and C) or 1 µM camptothecin (D, E, and F) for 6 hours to induce p53 expression. The cells were fixed, permeabilized, blocked and incubated with 1 µg/mL of the p53 antibody. Samples were further processed for fluorescence immunocytochemistry and co-stained with the DNA stain DAPI. Images of DAPI signals (A and D), p53 signals (B and E) and overlays of DAPI (artificially colored red for better contrast) and anti-p53 (artificially colored green) images (C and F) are shown.

Note that the p53 antibody specifically labels nuclei of camptothecin-treated cells.