Anti-p53 antibody [EPR17343]

• ICC/IF

Lab

Immunocytochemistry/ Immunofluorescence - Anti-p53 antibody [EPR17343] (AB179477)

ab179477 staining p53 in wild-type HAP1 cells (top panel) and p53 knockout HAP1 cells (bottom panel). The cells were fixed with methanol (5min), 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 ab179477 at 1/500 dilution and ab195889 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 Rabbit IgG (Alexa Fluor® 488) (ab150081) at 2 μg/ml (shown in green). Nuclear DNA was labelled in blue with DAPI.

• WB

Supplier Data

Western blot - Anti-p53 antibody [EPR17343] (AB179477)

Blocking and diluting buffer and concentration : 5% NFDM/TBST.

Negative control : HL-60 (p53 null), NCI-H1299 (p53 null).

The expression profile/ molecular weight observed is consistent with what has been described in the literature (PMID : 21941372, PMID : 21779513, PMID : 25996291).

In Western blot, Anti-GAPDH antibody [EPR16891] - Loading Control (ab181602) staining at 1/200000 dilution.

In Western blot, Anti-p53 antibody [DO-1] - ChIP Grade (ab1101) staining at 1/1000 dilution.

In Western blot, Anti-p53 antibody [EPR17343] (ab179477) staining at 1/1000 dilution.

All lanes:

Western blot - Anti-TP53 (phospho S376+S377+S392) antibody [RM1161] (<a href='/en-us/products/primary-antibodies/tp53-phospho-s376s377s392-antibody-rm1161-ab322465'>ab322465</a>) at 1/1000 dilution

Lane 1:

Untreated HCT 116 (human colorectal carcinoma epithelial cell) whole cell lysate at 20 µg

Lane 2:

HCT 116 treated with 20uM etoposide for 15 hours whole cell lysate at 20 µg

Lane 3:

Untreated A549 (human lung carcinoma epithelial cell) whole cell lysate at 20 µg

Lane 4:

A549 treated with 500nM doxorubicin for 24 hours whole cell lysate at 20 µg

Lane 5:

A431 (human epidermoid carcinoma epithelial cell) whole cell lysate at 20 µg

Lane 6:

HL-60 (human acute promyelocytic leukemia promyeloblast) whole cell lysate at 20 µg

Lane 7:

NCI-H1299 (human lung 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 1/100000 dilution

Observed band size: 53 kDa,26-48 kDa,36 kDa

false

Exposure time: 10s

• WB

Supplier Data

Western blot - Anti-p53 antibody [EPR17343] (AB179477)

Blocking/dilution buffer : 5% NFDM/TBST.

All lanes:

Western blot - Anti-p53 antibody [EPR17343] (ab179477) at 1/20000 dilution

All lanes:

T-47D (Human ductal breast epithelial carcinoma cell line) whole cell lysates at 20 µg

Secondary

All lanes:

Goat Anti-Rabbit IgG, (H+L),Peroxidase conjugated at 1/1000 dilution

Predicted band size: 43 kDa

Observed band size: 44 kDa,53 kDa

false

• WB

Lab

Western blot - Anti-p53 antibody [EPR17343] (AB179477)

Western blot : Anti-p53 antibody [EPR17343] ab179477 staining at 1/2000 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 [EPR17343] (ab179477) at 1/2000 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

• WB

Supplier Data

Western blot - Anti-p53 antibody [EPR17343] (AB179477)

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

Saos-2, PC-3 and HL-60 cells are p53 null cell lines.

All lanes:

Western blot - Anti-p53 antibody [EPR17343] (ab179477) at 1/2000 dilution

Lane 1:

HEK-293 (Human epithelial cells from embryonic kidney) whole cell lysates at 20 µg

Lane 2:

A431 (Human epidermoid carcinoma) whole cell lysates at 20 µg

Lane 3:

Saos-2 (Human osteosarcoma cell line) whole cell lysates at 20 µg

Lane 4:

HL-60 (Human promyelocytic leukemia cells) whole cell lysates at 20 µg

Lane 5:

PC-3 (Human prostate adenocarcinoma cell line) whole cell lysates at 20 µg

Secondary

All lanes:

Goat Anti-Rabbit IgG, (H+L),Peroxidase conjugated at 1/1000 dilution

Predicted band size: 43 kDa

Observed band size: 44 kDa

false

• WB

Lab

Western blot - Anti-p53 antibody [EPR17343] (AB179477)

Lane 1 : Wild-type HAP1 cell lysate (20 μg)

Lane 2 : p53 knockout HAP1 cell lysate (20 μg)

Lane 3 : A431 cell lysate (20 μg)

Lane 4 : Saos-2 cell lysate (20 μg)

Lanes 1 - 4 : Merged signal (red and green). Green - ab179477 observed at 53 kDa. Red - loading control, ab8226, observed at 42 kDa.

ab179477 was shown to specifically react with p53 in wild type HAP1 cells along with additional cross reactive bands. No band was observed with p53 knockout samples were used.

Wild-type and p53 knockout samples were subjected to SDS-PAGE. ab179477 and ab8226 (loading control to beta Actin) were diluted to 1/2000 and 1/1000 respectively and incubated overnight at 4°C. Blots were developed with Goat anti-Rabbit IgG H&L (IRDye^® 800CW) preadsorbed (ab216773) and Goat anti-Mouse IgG H&L (IRDye^® 680RD) preadsorbed (ab216776) secondary antibodies at 1/10 000 dilution for 1 h at room temperature before imaging.

All lanes:

Western blot - Anti-p53 antibody [EPR17343] (ab179477)

Predicted band size: 43 kDa

false

• WB

Lab

Western blot - Anti-p53 antibody [EPR17343] (AB179477)

All lanes:

Western blot - Anti-p53 antibody [EPR17343] (ab179477) at 1/2000 dilution

Lane 1:

Untreated HCT 116 (Human colorectal carcinoma) whole cell lysate

Lane 2:

HCT 116 (Human colorectal carcinoma) whole cell lysate with 0.5μM Doxorubicin for 24hours whole cell lysate

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: 53 kDa

false