ab90363 staining p53 in wild-type Hap1 cells (top panel) and p53 knockout Hap1 cells (bottom panel). The cells were fixed with 100% methanol (5 min), permeabilized with 0.1% Triton X-100 for 5 minutes and then blocked with 1% BSA/10% normal goat serum/0.3M glycine in 0.1% PBS-Tween for 1h. The cells were then incubated with ab90363 at 1/500 dilution and ab6046 (Rabbit polyclonal to beta Tubulin) at 1/1000 dilution overnight at +4°C, followed by a further incubation at room temperature for 1h with a goat secondary antibody to mouse IgG (Alexa Fluor® 488) (ab150117) at 2 μg/ml (shown in green) and a goat secondary antibody to rabbit IgG (Alexa Fluor® 594) (ab150080) at 2 μg/ml (shown in pseudo color red).  Nuclear DNA was labelled in blue with DAPI.

Image was taken with a high-content analysis system (Perkin Elmer, Operetta CLS™).

All lanes : Anti-p53 antibody [pAb122] (ab90363) at 1/2000 dilution

Lane 1 : HCT-116 cell lysate, untreated
Lane 2 : HCT-116 cell lysate + 10 µM CPT-11 for 24 hrs
Lane 3 : Negative control: HCT-116 cell lysate, untreated
Lane 4 : Negative control: HCT-116 cell lysate + 10 µM CPT-11 for 24 hrs

Lysates/proteins at 30 µg per lane.

Secondary
All lanes : HRP-conjugated rabbit anti-mouse polyclonal at 1/5000 dilution

Developed using the ECL technique.

Performed under reducing conditions.

Predicted band size: 44 kDa
Additional bands at: 130 kDa (possible non-specific binding), 35 kDa (possible non-specific binding), 55 kDa. We are unsure as to the identity of these extra bands.


Exposure time: 5 minutes

Blocked with 5% milk for 1 hour at 21^oC.

See Abreview

All lanes : Anti-p53 antibody [pAb122] (ab90363) at 1/250 dilution

Lane 1 : A431
Lane 2 : Rat brain tissue extract
Lane 3 : Mouse brain tissue extract

Developed using the ECL technique.

Predicted band size: 44 kDa