All lanes : Anti-p53 (acetyl K370) antibody [EPR17496] (ab183544) at 1/1000 dilution

Lane 1 : HepG2 (human hepatocellular carcinoma) treated with Etoposide 20uM and Trichostatin A 500 nM for 6 hours whole cell lysates
Lane 2 : HepG2 (human hepatocellular carcinoma) untreated whole cell lysates

Lysates/proteins at 10 µg per lane.

Secondary
All lanes : Goat Anti-Rabbit IgG, (H+L),Peroxidase conjugated at 1/1000 dilution

Predicted band size: 43 kDa
Observed band size: 53 kDa why is the actual band size different from the predicted?

Blocking/Dilution buffer: 5% NFDM/TBST

Immunofluorescent analysis of 4% paraformaldehyde-fixed, 0.1% Triton X-100 NIH/3T3 (Mouse embyro fibroblast cells) cells labeling p53 (acetyl K370) with ab183544 at 1/500 dilution, followed by Goat anti-rabbit Alexa Fluor® 488 IgG) (ab150077) secondary antibody at 1/400 dilution (green). Confocal image showing nuclear and weakly cytoplasm staining on NIH/3T3 cell line.
The expression increased after treatment with Trichostatin A (500 ng/ml) for 4 hours. The nuclear counter stain is DAPI (blue). Tubulin is detected with ab7291 (anti-Tubulin mouse mAb) at 1/500 dilution and ab150120 (goat anti-mouse AlexaFluor®594 secondary antibody) at 1/500 dilution (red).
The negative controls are as follows;
1. ab183544 at 1/500 dilution followed by ab150120 (goat anti-mouse AlexaFluor®594 secondary antibody) at 1/500 dilution.
2. ab7291 (anti-Tubulin mouse mAb) at 1/500 dilution followed by ab150077 (goat anti-rabbit Alexa Fluor®488 (IgG H&L) at 1/400 dilution.

[J Cell Biol. May 22, 2006; 173(4): 533–544.]

 

p53 (acetyl K370) was immunoprecipitated from 1mg of HepG2 (Human liver hepatocellular carcinoma) whole cell extract treated with Etoposide 20uM and Trichostatin A 500 nM for 6 hours with ab183544 at 1/100 dilution. Western blot was performed from the immunoprecipitate using ab183544 at 1/1000 dilution. Anti-Rabbit IgG (HRP), specific to the non-reduced form of IgG, was used as secondary antibody at 1/1500 dilution. Lane 1: HepG2 whole cell extract treated with Etoposide 20uM and Trichostatin A 500 nM for 6 hours. Lane 2: PBS instead of HepG2
Blocking and dilution buffer and concentration: 5% NFDM/TBST.

Flow Cytometry analysis of NIH/3T3 (mouse embryo) treated (Red)/untreated (Green) with 500ng/ml Trichostatin A for 4 hours with purified ab183544 at 1/150 dilution. The secondary antibody was Goat anti rabbit IgG (Alexa Fluor^® 488) at 1/2000 dilution. A Rabbit monoclonal IgG (Black) was used as the isotype control and cells without incubation with primary antibody and secondary antibody (Blue) were used as unlabeled control.

All lanes : Anti-p53 (acetyl K370) antibody [EPR17496] (ab183544) at 1/10000 dilution

Lane 1 : NIH/3T3 (mouse embryo) treated with Trichostatin A 500 nM for 4hr whole cell lysates
Lane 2 : NIH/3T3 (mouse embryo) untreated whole cell lysates

Lysates/proteins at 10 µg per lane.

Secondary
All lanes : Goat Anti-Rabbit IgG, (H+L),Peroxidase conjugated at 1/1000 dilution

Predicted band size: 43 kDa
Observed band size: 53 kDa why is the actual band size different from the predicted?

Blocking/Dilution buffer: 5% NFDM/TBST

All lanes : Anti-p53 (acetyl K370) antibody [EPR17496] (ab183544) at 1/1000 dilution

Lane 1 : C6 (rat glioma) treated with Trichostatin A 500 nM for 4hr whole cell lysates
Lane 2 : C6 (rat glioma) untreated whole cell lysates

Lysates/proteins at 10 µg per lane.

Secondary
All lanes : Goat Anti-Rabbit IgG, (H+L),Peroxidase conjugated at 1/1000 dilution

Predicted band size: 43 kDa
Observed band size: 53 kDa why is the actual band size different from the predicted?

Blocking/Dilution buffer: 5% NFDM/TBST