All lanes : Anti-p53 (phospho S392) antibody [EP155Y] (ab33889) at 1/1000 dilution (purified)

Lane 1 : RAW 264.7 (Mouse Abelson murine leukemia virus-induced tumor macrophage) whole cell lysates
Lane 2 : RAW 264.7 (Mouse Abelson murine leukemia virus-induced tumor macrophage) whole cell lysates, then the membrane was incubated with alkaline phosphatase.

Lysates/proteins at 15 µg per lane.

Secondary
All lanes : Goat Anti-Rabbit IgG H&L (HRP) (ab97051) at 1/20000 dilution

Predicted band size: 44 kDa

Blocking and diluting buffer: 5% NFDM/TBST.

Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) analysis of human lung carcinoma tissue sections labeling p53 with Purified ab33889 at 1:250 dilution. Heat mediated antigen retrieval was performed using ab93684 (Tris/EDTA buffer, pH 9.0). Tissue was counterstained with Hematoxylin. ImmunoHistoProbe one step HRP Polymer (ready to use) secondary antibody was used at 1:0 dilution. PBS instead of the primary antibody was used as the negative control.

All lanes : Anti-p53 (phospho S392) antibody [EP155Y] (ab33889) at 1/1000 dilution (purified)

Lane 1 : HEK-293 (Human embryonic kidney epithelial cell) whole cell lysates
Lane 2 : HEK-293 (Human embryonic kidney epithelial cell) whole cell lysates, then the membrane was incubated with alkaline phosphatase.

Lysates/proteins at 15 µg per lane.

Secondary
All lanes : Goat Anti-Rabbit IgG H&L (HRP) (ab97051) at 1/20000 dilution

Predicted band size: 44 kDa

Blocking and diluting buffer: 5% NFDM/TBST.

All lanes : Anti-p53 (phospho S392) antibody [EP155Y] (ab33889) at 1/1000 dilution (purified)

Lane 1 : PC-12 (Rat adrenal gland pheochromocytoma) whole cell lysates
Lane 2 : PC-12 (Rat adrenal gland pheochromocytoma) whole cell lysates, then the membrane was incubated with alkaline phosphatase.

Lysates/proteins at 15 µg per lane.

Secondary
All lanes : Goat Anti-Rabbit IgG H&L (HRP) (ab97051) at 1/20000 dilution

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

Blocking and diluting buffer: 5% NFDM/TBST

ab33889 (purified) at 1:20 dilution (0.6μg) immunoprecipitating p53 in 293T whole cell lysate.

Lane 1 (input): 293T (Human embryonic kidney epithelial cell) whole cell lysate, 10μg   
Lane 2 (+): ab33889 & 293T whole cell lysate
Lane 3 (-): Rabbit monoclonal IgG (ab172730) instead of ab33889 in 293T whole cell lysate.

For western blotting, VeriBlot for IP Detection Reagent (HRP) (ab131366) was used for detection at 1:1000 dilution.
Blocking and diluting buffer: 5% NFDM/TBST.

All lanes : Anti-p53 (phospho S392) antibody [EP155Y] (ab33889) at 1/2000 dilution (purified)

Lane 1 : A431 whole cell lysate
Lane 2 : A431 treated with 1µg/ml doxorubicin for 24 hours whole cell lysate
Lane 3 : A431 treated with 1µg/ml doxorubicin for 24 hours whole cell lysate, the membrane was incubated with alkaline phosphatase.

Lysates/proteins at 10 µg per lane.

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

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


Exposure time: 15 seconds

Blocking and dilution buffer: 5% NFDM/TBST.

Unpurified ab33889, at a 1/100 dilution, staining p53 in paraffin embedded human prostate adenocarcinoma tissue by Immunohistochemistry.

Dot blot analysis of p53 (pS392) peptide (Lane 1) and p53 non-phospho peptide (Lane 2) labelling p53 (pS392) with unpurified ab33889 at a dilution of 1/1000. ab97051 (Peroxidase conjugated goat anti-rabbit IgG (H+L)) was used as the secondary antibody at a dilution of 1/100000.

Blocking and dilution buffer: 5% NFDM/TBST.

Exposure time: 3 minutes.

All lanes : Anti-p53 (phospho S392) antibody [EP155Y] (ab33889) at 1/1000 dilution (unpurified)

Lane 1 : HEK293 whole cell lysate - untreated
Lane 2 : HEK293 whole cell lysate - treated with Alkaline Phosphatase

Lysates/proteins at 10 µg per lane.

Secondary
All lanes : Peroxidase-conjugated goat anti-rabbit IgG (H+L) at 1/1000 dilution

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


Exposure time: 15 seconds

Blocking and dilution buffer: 5% NFDM/TBST.

Lanes 1 and 5: Extract of Hek293T incubated with etoposide (7.5 µg).
Lanes 2 and 6: Lambda phosphatase (400 times-diluted)-treated extract of Hek293T incubated with etoposide (7.5 µg).
Lanes 3 and 7: Lambda phosphatase (100 times-diluted)-treated extract of Hek293T incubated with etoposide (7.5 µg).
Lanes 4 and 8: Lambda phosphatase (25 times-diluted)-treated extract of Hek293T incubated with etoposide (7.5 µg).

SDS PAGE performed under reducing conditions (100mM DTT, sample heated at 50°C).

Primary:
Lanes 1-4: Anti p53 (phosphoS392) antibody (ab33889, unpurified) at 1/2000 dilution.
Lanes 5-8: Anti p53 antibody (ab1101) at 1/2500 dilution.

Secondary:
Lanes 1-4: HRP-conjugated goat anti-rabbit IgG (H&L) at 1/10000.
Lanes 5-8: HRP-conjugated goat anti-mouse IgG (H&L) at 1/10000.

Blocked in 5% milk in PBS for 3 hours at room temperature.
Incubated with the primary antibody in 5% BSA + 50mM Tris pH 7.5 + 0.05% Tween-20 overnight at 4°C.
Incubated with the secondary antibody in blocking buffer for 2 hours at room temperature.

All lanes : Anti-p53 (phospho S392) antibody [EP155Y] (ab33889) at 1/500 dilution (unpurified)

Lane 1 : MCF7 cell lysate untreated.
Lane 2 : MCF7 cell lysate treated with 5 ug/ml Actinomycin for 3hrs.
Lane 3 : MCF7 cell lysate treated with 5 ug/ml Actinomycin for 6hrs.
Lane 4 : MCF7 cell lysate treated with 5 ug/ml Actinomycin for 18hrs.

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