Anti-p53 antibody [E47]

• ICC/IF

Lab

Immunocytochemistry/ Immunofluorescence - Anti-p53 antibody [E47] (AB32509)

ab32509 staining mutant p53 in wild-type Hap1 cells (top panel) and p53 knockout Hap1 cells (bottom panel). The cells were fixed with 4% paraformaldehyde (10 min), permeabilized with 0.1% PBS-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 overnight at 4°C with ab32509 at 0.2µg/ml and ab7291, Mouse monoclonal [DM1A] to alpha Tubulin - Loading Control. Cells were then incubated with ab150081, Goat polyclonal Secondary Antibody to Rabbit IgG - H&L (Alexa Fluor® 488), pre-adsorbed at 1/1000 dilution (shown in green) and ab150120, Goat polyclonal Secondary Antibody to Mouse IgG - H&L (Alexa Fluor® 594), pre-adsorbed at 1/1000 dilution (shown in pseudocolour red). Nuclear DNA was labelled with DAPI (shown in blue). Also suitable in cells fixed with 100% methanol (5 min). Image was acquired with a high-content analyser (Operetta CLS, Perkin Elmer) and a maximum intensity projection of confocal sections is shown.

• Flow Cyt (Intra)

Lab

Flow Cytometry (Intracellular) - Anti-p53 antibody [E47] (AB32509)

Intracellular Flow Cytometry analysis of HEK-293 (Human embryonic kidney epithelial cell) cells labeling p53 with purified ab32509 at 1/100 dilution (10 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. Isotype control - Rabbit monoclonal IgG (Black). Unlabeled control - Cells without incubation with primary antibody and secondary antibody (Blue).

• IHC-P

Unknown

Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-p53 antibody [E47] (AB32509)

Immunohistochemical analysis of paraffin-embedded human lung carcinoma sections labeling mutant p53 with ab32509 at 1/2000 dilution (0.49 μg/mL). Sections were counterstained with Hematoxylin. Goat Anti-Rabbit IgG H&L (HRP Polymer) was used as the secondary antibody. Heat mediated antigen retrieval was performed using ab93684 (Tris/EDTA buffer, pH 9.0).

Nuclear staining on human lung carcinoma.

• Flow Cyt (Intra)

Supplier Data

Flow Cytometry (Intracellular) - Anti-p53 antibody [E47] (AB32509)

Flow cytometry overlay histogram showing wild-type p53 in Hek-293 positive cells (left) and MCF7 negative cells (right) stained with ab32509 (red line). The cells were fixed with 4% paraformaldehyde (10 min) and then permeabilised 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 (ab32509) (1x 106 cells in 100μl at 0.2μg/ml (1/11865)) for 30min at 22°C. The secondary antibody Goat Anti-Rabbit IgG H&L (Alexa Fluor® 488) preadsorbed (ab150081) was incubated at 1/4000 for 30min at 22°C. Isotype control antibody (black line) Recombinant Rabbit IgG, monoclonal [EPR25A] - Isotype Control (ab172730) was used at the same concentration and conditions as the primary antibody. Unlabelled sample (blue line) was also used as a control. 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 Hek-293 fixed with 80% methanol (5 min) / permeabilised with 0.1% PBS-Triton X-100 for 15 min under the same conditions.

• Flow Cyt (Intra)

Supplier Data

Flow Cytometry (Intracellular) - Anti-p53 antibody [E47] (AB32509)

Flow cytometry overlay histogram showing mutant p53 in wild-type HAP1 cells (green line) and TP53 knockout HAP1 cells (red line) stained with ab32509. The cells were fixed with 4% paraformaldehyde (10 min) and then permeabilised 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 (ab32509) (1x 106 cells in 100μl at 0.2 μg/ml (1/550)) for 30min at 22°C. The secondary antibody Goat Anti-Rabbit IgG H&L (Alexa Fluor® 488) preadsorbed (ab150081) was incubated at 1/4000 for 30min at 22°C. Isotype control antibody Recombinant Rabbit IgG, monoclonal [EPR25A] - Isotype Control (ab172730) was 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 HAP1 fixed with 80% methanol (5 min) / permeabilised with 0.1% PBS-Triton X-100 for 15 min under the same conditions.

• Flow Cyt (Intra)

Supplier Data

Flow Cytometry (Intracellular) - Anti-p53 antibody [E47] (AB32509)

Flow cytometry overlay histogram showing mutant p53 in A-431 cells stained with ab32509 (red line). The cells were fixed with 4% paraformaldehyde (10 min) and then permeabilised with 0.1% PBS-Triton X-100 for 15 min. The cells were incubated in 1x PBS containing 10% normal goat serum to block non-specific protein-protein interaction followed by the antibody (ab32509) (1x 106 cells in 100μl at 0.2μg/ml (1/550)) for 30min at 22°C. The secondary antibody Goat Anti-Rabbit IgG H&L (Alexa Fluor® 488) preadsorbed (ab150081) was incubated at 1/4000 for 30min at 22°C. Isotype control antibody (black line) Recombinant Rabbit IgG, monoclonal [EPR25A] - Isotype Control (ab172730) was used at the same concentration and conditions as the primary antibody. Unlabelled sample (blue line) was also used as a control. 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 A-431 fixed with 80% methanol (5 min) / permeabilised with 0.1% PBS-Triton X-100 for 15 min under the same conditions.

• ICC/IF

Lab

Immunocytochemistry/ Immunofluorescence - Anti-p53 antibody [E47] (AB32509)

ab32509 staining mutant p53 in A431 cells. The cells were fixed with 100% methanol (5 min), permeabilized with 0.1% PBS-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 overnight at 4°C with ab32509 at 0.2µg/ml and ab7291, Mouse monoclonal [DM1A] to alpha Tubulin - Loading Control. Cells were then incubated with ab150081, Goat polyclonal Secondary Antibody to Rabbit IgG - H&L (Alexa Fluor® 488), pre-adsorbed at 1/1000 dilution (shown in green) and ab150120, Goat polyclonal Secondary Antibody to Mouse IgG - H&L (Alexa Fluor® 594), pre-adsorbed at 1/1000 dilution (shown in pseudocolour red). Nuclear DNA was labelled with DAPI (shown in blue). Also suitable in cells fixed with 4% paraformaldehyde (10 min). Image was acquired with a high-content analyser (Operetta CLS, Perkin Elmer) and a maximum intensity projection of confocal sections is shown.

• ICC/IF

Lab

Immunocytochemistry/ Immunofluorescence - Anti-p53 antibody [E47] (AB32509)

ab32509 staining wild-type p53 inMCF7 cells (a low expressing cell line). The cells were fixed with 100% methanol (5 min), permeabilized with 0.1% PBS-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 overnight at 4°C with ab32509 at 0.2µg/ml and ab7291, Mouse monoclonal [DM1A] to alpha Tubulin - Loading Control. Cells were then incubated with ab150081, Goat polyclonal Secondary Antibody to Rabbit IgG - H&L (Alexa Fluor® 488), pre-adsorbed at 1/1000 dilution (shown in green) and ab150120, Goat polyclonal Secondary Antibody to Mouse IgG - H&L (Alexa Fluor® 594), pre-adsorbed at 1/1000 dilution (shown in pseudocolour red). Nuclear DNA was labelled with DAPI (shown in blue). Also suitable in cells fixed with 4% paraformaldehyde (10 min). Image was acquired with a high-content analyser (Operetta CLS, Perkin Elmer) and a maximum intensity projection of confocal sections is shown.

• ICC/IF

Lab

Immunocytochemistry/ Immunofluorescence - Anti-p53 antibody [E47] (AB32509)

ab32509 staining wild-type p53 in Hek293 cells (a high expressing cell line). The cells were fixed with 100% methanol (5 min), permeabilized with 0.1% PBS-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 overnight at 4°C with ab32509 at 0.2µg/ml and ab7291, Mouse monoclonal [DM1A] to alpha Tubulin - Loading Control. Cells were then incubated with ab150081, Goat polyclonal Secondary Antibody to Rabbit IgG - H&L (Alexa Fluor® 488), pre-adsorbed at 1/1000 dilution (shown in green) and ab150120, Goat polyclonal Secondary Antibody to Mouse IgG - H&L (Alexa Fluor® 594), pre-adsorbed at 1/1000 dilution (shown in pseudocolour red). Nuclear DNA was labelled with DAPI (shown in blue). Also suitable in cells fixed with 4% paraformaldehyde (10 min). Image was acquired with a high-content analyser (Operetta CLS, Perkin Elmer) and a maximum intensity projection of confocal sections is shown.

• WB

Unknown

Western blot - Anti-p53 antibody [E47] (AB32509)

Blocking/Diluting buffer and concentration : 5% NFDM/TBST

Exposure time : 180 seconds

Lane 1-3 : Wildtype p53 cell lines

Lane 4-5 : Mutant p53 cell lines

Observed MW : 50kDa, 39kDa

All lanes:

Western blot - Anti-p53 antibody [E47] (ab32509) at 1/200 dilution

Lane 1:

A549 (Human lung carcinoma epithelial cell), Whole cell lysate at 20 µg

Lane 2:

HepG2 (Human hepatocellular carcinoma epithelial cell), Whole cell lysate at 20 µg

Lane 3:

MCF-7 (Human breast adenocarcinoma epithelial cell), Whole cell lysate at 20 µg

Lane 4:

T-47D (Human ductal breast epithelial tumor epithelial cell), Whole cell lysate at 20 µg

Lane 5:

A431 (Human epidermoid carcinoma epithelial cell), Whole cell lysate at 20 µg

Secondary

All lanes:

Western blot - Goat Anti-Rabbit IgG H&L (HRP) (<a href='/en-us/products/secondary-antibodies/goat-rabbit-igg-h-l-hrp-ab97051'>ab97051</a>) at 1/20000 dilution

Predicted band size: 43 kDa

false

• WB

Lab

Western blot - Anti-p53 antibody [E47] (AB32509)

False colour image of Western blot : Anti-p53 antibody [E47] staining at 1/1000 dilution, shown in green; Mouse anti-GAPDH antibody [6C5] (ab8245) loading control staining at 1/20000 dilution, shown in red. In Western blot, ab32509 was shown to bind specifically to p53. A band was observed at 50 kDa in wild-type HAP1 cell lysate with no signal observed at this size in tp53 knockout cell line. To generate this image, wild-type and tp53 knockout HAP1 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.

All lanes:

Western blot - Anti-p53 antibody [E47] (ab32509) at 1/1000 dilution

Lane 1:

Saos-2 cell lysate at 20 µg

Lane 2:

A431 cell lysate at 20 µg

Lane 3:

Wild-type HAP1 cell lysate at 20 µg

Lane 4:

TP53 knockout HAP1 cell lysate at 20 µg

Lane 5:

MCF7 cell lysate at 20 µg

Lane 6:

HEK-293T cell lysate at 20 µg

Secondary

Lanes 1 - 6:

Western blot - Goat anti-Mouse IgG H&L (IRDye® 680RD) preadsorbed (<a href='/en-us/products/secondary-antibodies/goat-mouse-igg-h-l-irdye-680rd-preadsorbed-ab216776'>ab216776</a>) at 1/20000 dilution

Lanes 1 - 6:

Western blot - Goat anti-Rabbit IgG H&L (IRDye® 800CW) preadsorbed (<a href='/en-us/products/secondary-antibodies/goat-rabbit-igg-h-l-irdye-800cw-preadsorbed-ab216773'>ab216773</a>) at 1/20000 dilution

Predicted band size: 43 kDa

Observed band size: 50 kDa

false

• IP

Lab

Immunoprecipitation - Anti-p53 antibody [E47] (AB32509)

Purified ab32509 at 1/50 dilution (2μg) immunoprecipitating p53 in HEK-293 whole cell lysate.
Lane 1 (input) : HEK-293 (Human embryonic kidney epithelial cell) whole cell lysate 10μg
Lane 2 (+) : ab32509 + HEK-293 whole cell lysate.
Lane 3 (-) : Rabbit monoclonal IgG (ab172730) instead of ab32509 in HEK-293 whole cell lysate.
VeriBlot for IP Detection Reagent (HRP) (ab131366) (1/1000 dilution) was used for Western blotting.
Blocking Buffer and concentration : 5% NFDM/TBST.
Diluting buffer and concentration : 5% NFDM/TBST.
Observed band size : 53 kDa

All lanes:

Immunoprecipitation - Anti-p53 antibody [E47] (ab32509)

Predicted band size: 43 kDa

false

• WB

CiteAb

Western blot - Anti-p53 antibody [E47] (AB32509)

p53 western blot using anti-p53 antibody [E47] ab32509. Publication image and figure legend from Yang, K., Wang, M., et al., 2016, Nat Commun, PubMed 27886181.

ab32509 was used in this publication in western blot. This may not be the same as the application(s) guaranteed by Abcam. For a full list of applications guaranteed by Abcam for ab32509 please see the product overview.

NPM1 translocation is required for p53 activation.(a,b) Endogenous NPM1 translocation and p53 accumulation cells under nucleolar stress with western blot validation. (a) Cells were pretreated with NAC (5 mM) for 4 h before Act.D (8 nM) treatment, or co-treated with DTT (5 mM) and Act.D (8 nM). Nucleolar line profiles of NPM1 in representative cells are shown in Supplementary Fig. 6a. (b) NPM1 was silenced with shRNA (shNPM1 5'-untranslated region (UTR)) before Act.D treatment or H2O2 (500 μM). (c) Immunofluorescence for endogenous p53 accumulation in NPM1-silenced cells (shNPM1 5'-UTR) that added back FLAG-NPM1 WT, C275S or WW mutants before treatment with Act.D. Line profiles of p53 in representative cells were showed in Supplementary Fig. 6c. (d) p53 accumulation after Act.D (left) or H2O2 (right) treatment in NPM1-silenced cells which were added back WT or C275S mutant of NPM1. Endogenous and exogenous FLAG-NPM1 were blotted with anti-NPM1 antibody. Nutlin-3 was used as a positive control for p53 inducer. (e) Relative mRNA level changes of p21 and PUMA after Act.D treatments in NPM1-silenced cells (shNPM1 5'-UTR) that added back FLAG-NPM1 WT or C275S mutants. Unpaired t-test. *p<0.05 or showed above the bars. Data were represented as mean±s.e.m. Scale bars, 10 μm. WW, W280/290A. All of the experiments were performed in U2OS cells.

false