Anti-p53 (phospho S15) antibody [EPR64(N)] - ChIP Grade

• ICC/IF

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Immunocytochemistry/ Immunofluorescence - Anti-p53 (phospho S15) antibody [EPR64(N)] - ChIP Grade (AB223868)

Immunofluorescent analysis of 4% PFA-fixed A431 (human epidermoid carcinoma cell line) cells labeling p53 (phospho S15) with ab223868 at 1/500 dilution, followed by Goat Anti-Rabbit IgG H&L (Alexa Fluor^® 488) (ab150077) secondary antibody at 1/1000 dilution (green). Confocal image showing increased nuclear staining on A431 cells treated with 1 μg/ml doxorubicin for 24 hours.

The nuclear counter stain is DAPI (blue). Tubulin is detected with 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

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Immunoprecipitation - Anti-p53 (phospho S15) antibody [EPR64(N)] - ChIP Grade (AB223868)

p53 (phospho S15) was immunoprecipitated from 0.35 mg of A431 (human epidermoid carcinoma cell line) whole cell lysate with ab223868 at 1/40 dilution. Western blot was performed from the immunoprecipitate using ab223868 at 1/1000 dilution. VeriBlot for IP Detection Reagent (HRP) (ab131366), was used for detection at 1/10000 dilution.

Lane 1 : A431 treated with 1 μg/ml doxorubicin for 24 hours, whole cell lysate 10 μg (Input).

Lane 2 : ab223868 IP in A431 treated with 1 μg/ml doxorubicin for 24 hours, whole cell lysate.

Lane 3 : Rabbit monoclonal IgG (ab172730) instead of ab223868 in A431 treated with 1 μg/ml doxorubicin for 24 hours, whole cell lysate.

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

Exposure time : 30 seconds.

All lanes:

Immunoprecipitation - Anti-p53 (phospho S15) antibody [EPR64(N)] - ChIP Grade (ab223868)

Predicted band size: 43 kDa

Observed band size: 53 kDa

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• ChIP

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ChIP - Anti-p53 (phospho S15) antibody [EPR64(N)] - ChIP Grade (AB223868)

Chromatin was prepared from HCT 116 (human colorectal carcinoma cell line) cells untreated or treated with 50 μM Etoposide for 6 hours according to the Abcam X-ChIP protocol. Cells were fixed with formaldehyde for 10 minutes. ChIP was performed with 25 mg of chromatin, 5 μg of ab223868 anti-p53 (phospho S15) (blue), and 20 μl of A/G sepharose beads slurry (10 μl of sepharose A beads + 10 μl of sepharose G beads). Rabbit normal IgG was added to the beads control (yellow). The immunoprecipitated DNA was quantified by real time PCR (SYBR approach).

• WB

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Western blot - Anti-p53 (phospho S15) antibody [EPR64(N)] - ChIP Grade (AB223868)

Blocking/Dilution buffer : 2% BSA/TBST.

All lanes:

Western blot - Anti-p53 (phospho S15) antibody [EPR64(N)] - ChIP Grade (ab223868) at 1/5000 dilution

Lane 1:

Untreated A431 (human epidermoid carcinoma cell line), whole cell lysate at 10 µg

Lane 2:

A431 (human epidermoid carcinoma cell line) treated with 1 μg/ml doxorubicin for 24 hours, 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: 1s

• ChIC/CUT&RUN-seq

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ChIC/CUT&RUN sequencing - Anti-p53 (phospho S15) antibody [EPR64(N)] - ChIP Grade (AB223868)

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 ab223868 [EPR64(N]. 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.

• Dot

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Dot Blot - Anti-p53 (phospho S15) antibody [EPR64(N)] - ChIP Grade (AB223868)

Dot blot analysis of p53 (phospho S15) labeled with ab223868 at 1/1000 dilution.

Lane 1 : p53 (phospho S15) peptide;

Lane 2 : p53 non-phospho peptide;

Goat Anti-Rabbit IgG H&L (HRP) (ab97051) at 1/100000 dilution was used as secondary antibody.

Blocking/Dilution buffer : 5% NFDM/TBST.

Exposure time : 3 minutes.