Anti-p53 antibody [EPR20416-120]

• Flow Cyt (Intra)

Unknown

Flow Cytometry (Intracellular) - Anti-p53 antibody [EPR20416-120] (AB246550)

Intracellular flow cytometric analysis of 4% paraformaldehyde-fixed, 90% methanol-permeabilized LLC1 (mouse lung carcinoma cell line) cell line labeling p53 with ab246550 at 1/60 dilution (red) compared with a Rabbit IgG, monoclonal [EPR25A] - Isotype Control (ab172730) (black) and an unlabeled control (cells without incubation with primary antibody and secondary antibody) (blue). Goat Anti-Rabbit IgG H&L (Alexa Fluor^® 488) (ab150077) at 1/2000 dilution was used as the secondary antibody.

• ICC/IF

Unknown

Immunocytochemistry/ Immunofluorescence - Anti-p53 antibody [EPR20416-120] (AB246550)

Immunofluorescent analysis of 4% paraformaldehyde-fixed, 0.1% Triton X-100 permeabilized LLC1 (mouse lung carcinoma cell line) cells labeling p53 with ab246550 at 1/100 dilution followed by a Goat Anti-Rabbit IgG H&L (Alexa Fluor^® 488) (ab150077) secondary antibody at 1/1000 dilution (green). Confocal image showing nuclear staining in LLC1 cells is observed.

The nuclear counter stain is DAPI (blue). Tubulin is detected with an Anti-alpha Tubulin antibody [DM1A] - Microtubule Marker (Alexa Fluor^® 594) (ab195889) (red) at 1/200 dilution.

Secondary antibody only control : Used PBS instead of primary antibody, secondary antibody is Goat Anti-Rabbit IgG H&L (Alexa Fluor^® 488) (ab150077) secondary antibody at 1/1000 dilution.

• IP

Unknown

Immunoprecipitation - Anti-p53 antibody [EPR20416-120] (AB246550)

p53 was immunoprecipitated from 0.35 mg of LLC1 (mouse lung carcinoma cell line) whole cell lysate with ab246550 at 1/30 dilution. Western blot was performed from the immunoprecipitate using ab246550 at 1/2000 dilution. VeriBlot for IP Detection Reagent (HRP) (ab131366), was used for detection at 1/5000 dilution.

Lane 1 : LLC1 whole cell lysate 10 μg (Input).

Lane 2 : ab246550 IP in LLC1 whole cell lysate.

Lane 3 : Rabbit monoclonal IgG (ab172730) instead of ab246550 in LLC1 whole cell lysate.

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

Exposure time : 10 seconds.

All lanes:

Immunoprecipitation - Anti-p53 antibody [EPR20416-120] (ab246550)

Predicted band size: 43 kDa

Observed band size: 53 kDa

false

• ChIC/CUT&RUN-seq

Supplier Data

ChIC/CUT&RUN sequencing - Anti-p53 antibody [EPR20416-120] (AB246550)

ChIC/CUT&RUN was performed using a pAG-MNAse at a final concentration of 700 ng/mL, 2.5 x 10^5 U2OS cells treated with Etoposide (5μM 18h) and 5 µg of ab246550 [EPR20416-120]. The resulting DNA was sequenced on the Illumina NovaSeq 6000 to a depth of 10 million reads. The negative IgG control ab172730 is also shown. Additional screenshots of mapped reads can be found in the Protocol booklet in the Product Protocol section. The University of Geneva owns patents relevant to ChIC (Chromatin Immuno-Cleavage) methods.

• WB

Supplier Data

Western blot - Anti-p53 antibody [EPR20416-120] (AB246550)

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

The expression of acetyl p53 is upregulated in response to doxorubicin and TSA treatment (PMID : 15123817).

The identity of the lower MW band at approximately 15 kDa is unknown.

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

All lanes:

Western blot - Anti-p53 (acetyl K305 + K370 + K373) antibody [RM1200] (<a href='/en-us/products/primary-antibodies/p53-acetyl-k305-k370-k373-antibody-rm1200-ab321819'>ab321819</a>) at 1/1000 dilution

Lane 1:

NIH/3T3 (mouse embryonic fibroblast) whole cell lysate at 20 µg

Lane 2:

NIH/3T3 treated with 0.5µM Doxorubicin (<a href='/en-us/products/biochemicals/doxorubicin-hydrochloride-topoisomerase-ii-inhibitor-ab120629'>ab120629</a>) for 24 hours whole cell lysate at 20 µg

Lane 3:

NIH/3T3 treated with 0.5µM Doxorubicin and 500ng/ml TSA(Trichostatin A) for 24 hours 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,36 kDa

false

Exposure time: 180s

• WB

Unknown

Western blot - Anti-p53 antibody [EPR20416-120] (AB246550)

Blocking/Dilution buffer : 5% NFDM/TBST.

The molecular weight observed is consistent with what has been described in the literature (PMID : 27702985).

All lanes:

Western blot - Anti-p53 antibody [EPR20416-120] (ab246550) at 1/1000 dilution

All lanes:

LLC1 (mouse lung carcinoma cell line) whole cell lysate at 10 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

Predicted band size: 43 kDa

Observed band size: 53 kDa

true

Exposure time: 92s

• WB

Lab

Western blot - Anti-p53 antibody [EPR20416-120] (AB246550)

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

In Western blot, Anti-GAPDH antibody [EPR16891] - Loading Control (ab181602) 1 : 1,000,000 dilution

Low or negative sample : normal brain (PMID : 32778161); normal liver (PMID : 10949934); NIH/3T3 (PMID : 10628835).

All lanes:

Western blot - Anti-p53 antibody [EPR20416-120] (ab246550) at 1/1000 dilution

Lane 1:

Mouse spleen tissue lysate at 20 µg

Lane 2:

Mouse thymus tissue lysate at 20 µg

Lane 3:

Mouse brain tissue lysate at 20 µg

Lane 4:

Mouse liver tissue lysate at 20 µg

Lane 5:

NIH/3T3 (mouse embryonic fibroblast) 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: 53 kDa

Observed band size: 53 kDa

false

Exposure time: 180s