Anti-p53 antibody [E47] - BSA and Azide free

• Flow Cyt (Intra)

Supplier Data

Flow Cytometry (Intracellular) - Anti-p53 antibody [E47] - BSA and Azide free (AB247264)

This data was developed using the same antibody clone in a different buffer formulation containing PBS and sodium azide (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] - BSA and Azide free (AB247264)

This data was developed using the same antibody clone in a different buffer formulation containing PBS and sodium azide (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] - BSA and Azide free (AB247264)

This data was developed using the same antibody clone in a different buffer formulation containing PBS and sodium azide (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] - BSA and Azide free (AB247264)

This data was developed using ab32509, the same antibody clone in a different buffer formulation.Intracellular Flow Cytometry analysis of HEK-293 (Human embryonic kidney epithelial cell) cells labeling p53 with purified ab32509 at 1/100 dilution (10μg/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] - BSA and Azide free (AB247264)

This data was developed using ab32509, the same antibody clone in a different buffer formulation.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).

• ICC/IF

Lab

Immunocytochemistry/ Immunofluorescence - Anti-p53 antibody [E47] - BSA and Azide free (AB247264)

This data was developed using the same antibody clone in a different buffer formulation containing PBS and sodium azide (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] - BSA and Azide free (AB247264)

This data was developed using the same antibody clone in a different buffer formulation containing PBS and sodium azide (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.

• Flow Cyt (Intra)

Supplier Data

Flow Cytometry (Intracellular) - Anti-p53 antibody [E47] - BSA and Azide free (AB247264)

This data was developed using the same antibody clone in a different buffer formulation containing PBS and sodium azide (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] - BSA and Azide free (AB247264)

This data was developed using the same antibody clone in a different buffer formulation containing PBS and sodium azide (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.

• IP

Lab

Immunoprecipitation - Anti-p53 antibody [E47] - BSA and Azide free (AB247264)

This data was developed using ab32509, the same antibody clone in a different buffer formulation.
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] (<a href='/en-us/products/primary-antibodies/p53-antibody-e47-ab32509'>ab32509</a>)

Predicted band size: 43 kDa

false

• WB

Lab

Western blot - Anti-p53 antibody [E47] - BSA and Azide free (AB247264)

This data was developed using the same antibody clone in a different buffer formulation containing PBS and sodium azide (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] (<a href='/en-us/products/primary-antibodies/p53-antibody-e47-ab32509'>ab32509</a>) 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

• WB

Unknown

Western blot - Anti-p53 antibody [E47] - BSA and Azide free (AB247264)

This data was developed using ab32509, the same antibody clone in a different buffer formulation.

Blocking and dilution buffer : 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] - BSA and Azide free (ab247264) 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