Predicted band size : 43.6 kDa

Lanes 1, 5 and 9: Wild-type HAP1 cell lysate (20 µg)
Lanes 2, 6 and 10: p53 knockout HAP1 cell lysate (20 µg)
Lanes 3, 7 and 11: A431 cell lysate (20 µg)
Lanes 4, 8 and 12: Saos-2 cell lysate (20 µg)
Lanes 1, 2, 3 and 4: Green signal from target – ab1101 observed at 53 kDa
Lanes 5, 6, 7 and 8: Red signal from loading control – ab181602 observed at 37 kDa
Lanes 9, 10, 11 and 12: Merged (red and green) signal

Anti-p53 antibody [DO-1] (ab1101) was shown to specifically react with p53 when p53 knockout samples were used. Wild-type and p53 knockout samples were subjected to SDS-PAGE. ab1101 and ab181602 (loading control to GAPDH) were diluted 1/1000 and 1/10000 respectively and incubated overnight at 4ºC. Blots were developed with Goat anti-Mouse IgG H&L (IRDye^® 800CW) preadsorbed (ab216772) and Goat Anti-Rabbit IgG H&L (IRDye^® 680RD) preadsorbed (ab216777) secondary antibodies at 1/10000 dilution for 1 h at room temperature before imaging.

ab32049 showing positive staining in urinary bladder carcinoma tissue.

All lanes : Anti-p53 (phospho S20) antibody [EPR2156(2)] (ab157454) at 1/1000 dilution

Lane 1 : T47D (human mammary gland ductal carcinoma) treated with Etoposide whole cell lysate
Lane 2 : Untreated T47D (human mammary gland ductal carcinoma) whole cell lysate

Lysates/proteins at 10 µg per lane.

Secondary
Goat anti-rabbit IgG, (H+L), peroxidase conjugated 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 : 1 minute

Blocking & dilution buffer: 5% NFDM/TBST

IHC image of ab75754 staining p53 (acetyl K382) in human breast adenocarcinoma formalin fixed paraffin embedded tissue sections*, performed on a Leica Bond. The section was pre-treated using heat mediated antigen retrieval with sodium citrate buffer (pH6, epitope retrieval solution 1) for 20 mins. The section was then incubated with ab75754, 1μg/ml working concentration, for 15 mins at room temperature and detected using an HRP conjugated compact polymer system. DAB was used as the chromogen. The section was then counterstained with haematoxylin and mounted with DPX. No primary antibody was used in the secondary only control (shown on the inset).
For other IHC staining systems (automated and non-automated) customers should optimize variable parameters such as antigen retrieval conditions, primary antibody concentration and antibody incubation times.
*Tissue obtained from the Human Research Tissue Bank, supported by the NIHR Cambridge Biomedical Research Centre

All lanes : Anti-p53 (phospho S46) antibody [EP42Y] (ab76242) at 1/5000 dilution

Lane 1 : Untreated HepG2 whole cell lysate
Lane 2 : HepG2 whole cell lysate, treated with etoposide
Lane 3 : HepG2 whole cell lysate, treated with etoposide, then the membrane was treated with alkaline phosphatase

Lysates/proteins at 10 µg per lane.

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

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


Exposure time : 1 minute

Blocking buffer: 2% BSA/TBST
Dilution buffer: 2% BSA/TBST

Ab33889, at a 1/100 dilution, staining p53 in paraffin embedded human prostate adenocarcinoma tissue by Immunohistochemistry.