All lanes : Anti-p53 antibody [X77] (ab16465) at 5 µg/ml

Lane 1 : HAP1 wild-type cell lysate
Lane 2 : HAP1 TP53 knockout cell lysate
Lane 3 : A431 cell lysate

Lysates/proteins at 20 µg per lane.

Performed under reducing conditions.

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

Lanes 1 - 3: Merged signal (red and green). Green - ab16465 observed at 53 kDa. Red - loading control, ab8245 (Mouse anti-GAPDH antibody [6C5]) observed at 37kDa.

ab16465 was shown to react with TP53 in HAP1 wild-type cells in Western blot. Loss of signal was observed when TP53 knockout sample was used. HAP1 wild-type and TP53 knockout cell lysates were subjected to SDS-PAGE. Membranes were blocked in 3% Milk in TBS-T (0.1% Tween®) before incubation with ab16465 and ab8245 (Mouse anti-GAPDH antibody [6C5]) overnight at 4°C at 5 µg/ml and a 1 in 20000 dilution respectively. Blots were developed with Goat anti-Mouse IgG H&L (IRDye® 800CW) preabsorbed (ab216772) and Goat anti-Rabbit IgG H&L (IRDye® 680RD) preabsorbed (ab216777) secondary antibodies at 1 in 20000 dilution for 1 hour at room temperature before imaging.

ICC/IF image of ab16465 stained HepG2 cells. The cells were 4% PFA fixed (10 min) and then incubated in 1%BSA / 10% normal goat serum / 0.3M glycine in 0.1% PBS-Tween for 1h to permeabilise the cells and block non-specific protein-protein interactions. The cells were then incubated with the antibody (ab16465, 1µg/ml) overnight at +4°C. The secondary antibody (green) was Alexa Fluor^® 488 goat anti-mouse IgG (H+L) used at a 1/1000 dilution for 1h. Alexa Fluor^® 594 WGA was used to label plasma membranes (red) at a 1/200 dilution for 1h. DAPI was used to stain the cell nuclei (blue) at a concentration of 1.43µM.

Western blot analysis using ab16465 at 10 µg/ml on HCT116 cell lysate (1), HCT116 cell lysate activated with adriamycin (2), p21-/- cell lysate (3), P21-/- cell lysate activated with ADR (4) and p53-/- activated with ADR. ADR activates p53 in cells.