Anti-p53 antibody [E26] - BSA and Azide free

• IHC-P

Lab

Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-p53 antibody [E26] - BSA and Azide free (AB225531)

This data was developed using ab32389, the same antibody clone in a different buffer formulation.
Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) analysis of human endometrium cancer tissue sections labeling 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. PBS instead of the primary antibody was used as the negative control.

• ICC/IF

Lab

Immunocytochemistry/ Immunofluorescence - Anti-p53 antibody [E26] - BSA and Azide free (AB225531)

This data was developed using the same antibody clone in a different buffer formulation containing PBS, BSA, glycerol, and sodium azide (ab32389).

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™).

• ICC/IF

Lab

Immunocytochemistry/ Immunofluorescence - Anti-p53 antibody [E26] - BSA and Azide free (AB225531)

This data was developed using ab32389, the same antibody clone in a different buffer formulation.

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.

• Flow Cyt

Supplier Data

Flow Cytometry - Anti-p53 antibody [E26] - BSA and Azide free (AB225531)

This data was developed using ab32389 the same antibody clone in a different buffer formulation.

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.

• WB

Lab

Western blot - Anti-p53 antibody [E26] - BSA and Azide free (AB225531)

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.

All lanes:

Western blot - Anti-p53 antibody [E26] (<a href='/en-us/products/primary-antibodies/p53-antibody-e26-ab32389'>ab32389</a>) at 1/1000 dilution

Lane 1:

Wild-type A549 cell lysate at 20 µg

Lane 2:

TP53 knockout A549 cell lysate at 20 µg

Lane 2:

Western blot - Human TP53 (p53) knockout Jurkat cell line (<a href='/en-us/products/cell-lines/human-tp53-p53-knockout-jurkat-cell-line-ab276112'>ab276112</a>)

Lane 3:

Wild-type Jurkat cell lysate at 20 µg

Lane 4:

TP53 knockout Jurkat cell lysate at 20 µg

Lane 5:

A431 cell lysate at 20 µg

Lane 6:

Saos-2 cell lysate at 20 µg

Predicted band size: 43 kDa

Observed band size: 49 kDa

false

• WB

Lab

Western blot - Anti-p53 antibody [E26] - BSA and Azide free (AB225531)

This WB data was generated using the same antibody clone in a different buffer formulation (ab32389).

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

All lanes:

Western blot - Anti-p53 antibody [E26] (<a href='/en-us/products/primary-antibodies/p53-antibody-e26-ab32389'>ab32389</a>) at 1/10000 dilution

All lanes:

HEK-293 (Human embryonic kidney epithelial cell) whole cell lysate at 15 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

Observed band size: 46 kDa,53 kDa

false

• WB

Unknown

Western blot - Anti-p53 antibody [E26] - BSA and Azide free (AB225531)

This WB data was generated using the same anti-p53 antibody clone [E26] in a different buffer formulation (cat# ab32389).

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 cells as signal was lost in p53 knockout cells. 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.

All lanes:

Western blot - Anti-p53 antibody [E26] (<a href='/en-us/products/primary-antibodies/p53-antibody-e26-ab32389'>ab32389</a>)

Predicted band size: 43 kDa

false

• WB

Lab

Western blot - Anti-p53 antibody [E26] - BSA and Azide free (AB225531)

This data was developed using the same antibody clone in a different buffer formulation containing PBS, BSA, glycerol, and sodium azide (ab32389).

Western blot : Anti-p53 antibody [E26] ab32389 staining at 1/1000 dilution, shown in green; Mouse anti-CANX ab238078 loading control staining at 1/20000 dilution, shown in magenta. A band was observed at 50 kDa in Wild-type MCF7 cell lysates with no signal observed at this size in TP53 knockout MCF7 cell line. To generate this image, samples were run on an SDS-PAGE gel then transferred onto a nitrocellulose membrane. Membranes were blocked in 5pc 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 800CW and Goat anti-Mouse 680RD at 1/20,000 dilution.

All lanes:

Western blot - Anti-p53 antibody [E26] (<a href='/en-us/products/primary-antibodies/p53-antibody-e26-ab32389'>ab32389</a>) at 1/1000 dilution

Lane 1:

Wild-type MCF7 whole cell lysate at 20 µg

Lane 2:

Western blot - Human TP53 knockout MCF7 cell line (<a href='/en-us/products/cell-lines/human-tp53-knockout-mcf7-cell-line-ab286440'>ab286440</a>) at 20 µg

Lane 3:

Wild-type A549 whole cell lysate at 20 µg

Lane 4:

TP53 knockout A549 whole cell lysate at 20 µg

Secondary

All lanes:

Goat anti-Rabbit 800CW & Goat anti-Mouse 680RD at 1/20000 dilution

Predicted band size: 50 kDa

Observed band size: 50 kDa

false