All lanes : 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
Lane 2 : A431 (human epidermoid carcinoma cell line) treated with 1 µg/ml doxorubicin for 24 hours, whole cell lysate

Lysates/proteins at 10 µg per lane.

Secondary
All lanes : Goat Anti-Rabbit IgG H&L (HRP) (ab97051) at 1/100000 dilution

Developed using the ECL technique.

Predicted band size: 44 kDa
Observed band size: 53 kDa why is the actual band size different from the predicted?


Exposure time: 1 second

Blocking/Dilution buffer: 2% BSA/TBST.

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.

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.

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.

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).