All lanes : Anti-p53 antibody [PAb 240] (ab26) at 5 µg/ml

Lane 1 : Wild-type HCT116 cell lysate at 30 µg
Lane 2 : Wild-type HCT116 + irinotecan (10 µM, 24 hours) cell lysate at 30 µg
Lane 3 : p53 knockout HCT116 cell lysate at 30 µg
Lane 4 : p53 knockout HCT116 + irinotecan (10 µM, 24 hours) cell lysate at 30 µg
Lane 5 : A431 cell lysate (positive control) at 20 µg
Lane 6 : Saos-2 cell lysate (negative control) at 20 µg

Performed under reducing conditions.

Predicted band size: 53 kDa
Observed band size: 53 kDa

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.

Anti-p53 antibody [PAb 240] (ab26) at 1 µg/ml + HeLa (Human epithelial carcinoma cell line) Whole Cell Lysate at 10 µg

Secondary
Goat Anti-Mouse IgG H&L (HRP) preadsorbed (ab97040) at 1/50000 dilution

Developed using the ECL technique.

Performed under reducing conditions.

Predicted band size: 53 kDa
Observed band size: 53 kDa


Exposure time: 8 minutes

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

All lanes : Anti-p53 antibody [PAb 240] (ab26) at 5 µg/ml

Lane 1 : HeLa (Human epithelial carcinoma cell line) Whole Cell Lysate
Lane 2 : Hela Whole Cell Lysate - Bleomycin Treated (40U/ml)

Lysates/proteins at 20 µg per lane.

Secondary
All lanes : Goat Anti-Mouse IgG H&L (HRP) preadsorbed (ab97040) at 1/5000 dilution

Developed using the ECL technique.

Performed under reducing conditions.

Predicted band size: 53 kDa


Exposure time: 4 minutes

Lanes 1-2 : Anti-p53 antibody [PAb 240] (ab26) at 1 µg/ml
Lanes 3-4 : Anti-p53 antibody [PAb 240] (ab26) at 5 µg/ml

All lanes : HeLa (Human epithelial carcinoma cell line) Whole Cell Lysate

Lysates/proteins at 20 µg per lane.

Secondary
All lanes : Goat polyclonal to Mouse IgG - H&L - Pre-Adsorbed (HRP) at 1/5000 dilution

Developed using the ECL technique.

Performed under reducing conditions.

Predicted band size: 53 kDa


Exposure time: 4 minutes

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.

All lanes : Anti-p53 antibody [PAb 240] (ab26) at 1/2000 dilution

Lane 1 : Human breast cancer cell-line, MCF7 cells (p53 WT), whole cell lysate
Lane 2 : Human breast cancer cell-line, MDA231 cells (p53 Mutant), whole cell lysate

Lysates/proteins at 20 µg per lane.

Secondary
All lanes : HRP conjugated donkey anti-mouse antibody at 1/5000 dilution

Developed using the ECL technique.

Performed under reducing conditions.

Predicted band size: 53 kDa
Observed band size: 53 kDa
Additional bands at: 72 kDa (possible non-specific binding)


Exposure time: 10 seconds

See Abreview

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.