Anti-p53 antibody [PAb 240] (ab26)

Lanes 1-6: Merged (red and green) signal.

Ab26 was shown to specifically react with p53 in wild type HCT116 cells treated with irinotecan. No band was observed in p53 knockout HCT116 cells. Wild-type and p53 knockout samples, positive and negative controls were subjected to SDS-PAGE. Ab26 and ab181602(loading control to GAPDH) were diluted 5 μg/mL and 1/10,000 respectively and incubated overnight at 4°C. Blots were developed with goat anti-rabbit IgG (H + L) and goat anti-mouse IgG (H + L) secondary antibodies at 1/10,000 dilution for 1 h at room temperature before imaging.

Wild-type and p53 knockout HCT116 cell lysates were kindly provided by a collaborator.

ab26 stained in A431 cells. Cells were fixed with 4% paraformaldehyde (10min) at room temperature and incubated with PBS containing 10% goat serum, 0.3 M glycine, 1% BSA and 0.1% triton for 1h at room temperature to permeabilise the cells and block non-specific protein-protein interactions. The cells were then incubated with ab26 at 1µg/ml and ab6046 (Rabbit polyclonal to beta tubulin) at 1ug/ml overnight at +4°C. The secondary antibodies were ab150177 (colored green) used at 1 ug/ml and ab150087 (pseudo-colored red) used at 2ug/ml for 1hour at room temperature. DAPI was used to stain the cell nuclei (colored blue) at a concentration of 1.43µM for 1hour at room temperature.

Primary: All Lanes: Anti-p53 antibody (ab26) at 5 µg/mL. Lane 1: MW marker. Lane 2: NIH/3T3 cells treated with vehicle for 24 hours. Lane 3: NIH/3T3 cells treated with 1 µM doxorubicin for 24 hours Secondary: All Lanes: HRP-conjugated VeriBlot anti-Mouse IgG (ab131368) 1:1000. Lysates at 20 µg/lane. Performed under denaturing conditions. Developed using ECL technique. Blocking buffer: 5% milk in PBS.

Lanes 1-2: 1% BSA blocking buffer

Lanes 3-4: 3% Milk blocking buffer

We recommend using 3% milk as the blocking agent for Western blot.

This blot was produced using a 4-12% Bis-tris gel under the MOPS buffer system. The gel was run at 200V for 50 minutes before being transferred onto a Nitrocellulose membrane at 30V for 70 minutes. The membrane was then blocked for an hour using 2% Bovine Serum Albumin before being incubated with ab26 overnight at 4°C. Antibody binding was detected using an anti-mouse HRP secondary antibody, and visualised using ECL development solution ab133406

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p53 was immunoprecipitated from 7x10⁶ HCT116 (human colon carcinoma cell line) cells with ab26 at 1/150 dilution. Western blot was performed from the immunoprecipitate using anti-p53 antibody. Donkey Anti-Mouse IgG H&L (Alexa Fluor® 750) preadsorbed (ab175739) was used as secondary antibody at 1/5000 dilution.

Lane 1: HCT116 whole cell lysate 10 µg (Input).

Lane 2: ab207799 IP in etoposide treated HCT116 whole cell lysate.

Lane 3: ab207799 IP in etoposide treated HCT116 p53-/- whole cell lysate (negative control).