Recombinant Anti-p53 antibody [E26] (ab32389)

False colour image of Western blot: Anti-p53 antibody [E26] staining at 1/1000 dilution, shown in green; Mouse anti-Alpha Tubulin [DM1A] (ab7291) loading control staining at 1/20000 dilution, shown in red. In Western blot, ab32389 was shown to bind specifically to p53. A band was observed at 49 kDa in wild-type A549 cell lysates with no signal observed at this size in tp53 knockout cell line ab276092 (knockout cell lysate ab282999). To generate this image, wild-type and tp53 knockout A549 cell lysates were analysed. First, samples were run on an SDS-PAGE gel then transferred onto a nitrocellulose membrane. Membranes were blocked in 3% milk in TBS-0.1 % Tween^® 20 (TBS-T) before incubation with primary antibodies overnight at 4°C. Blots were washed four times in TBS-T, incubated with secondary antibodies for 1 h at room temperature, washed again four times then imaged. Secondary antibodies used were Goat anti-Rabbit IgG H&L (IRDye^® 800CW) preabsorbed (ab216773) and Goat anti-Mouse IgG H&L (IRDye^® 680RD) preabsorbed (ab216776) at 1/20000 dilution.

Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) analysis of human endometrium cancer tissue sections labelling p53 with purified ab32389 at 1/5000 dilution (0.09 µg/ml). Heat mediated antigen retrieval was performed using Heat mediated antigen retrieval using Bond™ Epitope Retrieval Solution 2 (pH 9.0) . Tissue was counterstained with Hematoxylin. Rabbit specific IHC polymer detection kit HRP/DAB (ab209101) secondary antibody was used at 1/0 dilution. PBS instead of the primary antibody was used as the negative control. The immunostaining was performed on a Leica Biosystems BOND® RX instrument.

Lane 1: Wild type HAP1 whole cell lysate (20 µg)
Lane 2: p53 knockout HAP1 whole cell lysate (20 µg)
Lane 3: HEK293 whole cell lysate (20 µg)
Lane 4: A431 whole cell lysate (20 µg)

Lanes 1 - 4: Merged signal (red and green). Green - ab32389 observed at 50 kDa. Red - loading control, ab9484, observed at 37 kDa.

ab32389 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. ab32389 and ab9484 (Mouse anti-GAPDH loading control) were incubated overnight at 4°C at 1/1000 dilution and 1/20000 dilution respectively. Blots were developed with Goat anti-Rabbit IgG H&L (IRDye^® 800CW) preabsorbed ab216773 and Goat anti-Mouse IgG H&L (IRDye^® 680RD) preabsorbed ab216776 secondary antibodies at 1/20000 dilution for 1 hour at room temperature before imaging.

ab32389 staining p53 in wild-type Hap1 cells (top panel) and p53 knockout Hap1 cells (bottom panel). The cells were fixed with 100% methanol (5 min), 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 ab32389 at 1µg/ml concentration 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™).

Flow cytometric analysis of 4% Paraformaldehyde fixed 90% Methanol permeabilized HEK-293 (Human embryonic kidney epithelial cell) cells labelling p53 with ab32389 at 1/500 dilution (1 µg/ml) compared with a Rabbit monoclonal IgG (Black) isotype control and an unlabelled control (cells without incubation with primary antibody and secondary antibody) (Blue). A Goat anti rabbit IgG (Alexa Fluor® 488, ab150077) at 1/2000 was used as the secondary antibody.

The isoforms of p53 produced by alternative splicing of the intron 9 have been described by the literatures (PMID: 16131611, 29235495 and 22647703).

Immunocytochemistry analysis of A431 (Human epidermoid carcinoma epithelial cell) cells labeling p53 with purified ab32389 at 1/50 dilution (8.7 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.