Anti-p53 p-Ser46 (human) antibody, mouse monoclonal (#36)
Product code71-115Size

100 µgStorage-20℃

Concentration

1.0 mg/ml

Buffer

PBS- with 50% glycerol

PurityPurified IgG fraction with protein A from hybridoma cell culture medium.

ImmunogenSynthetic peptide containing phospho-Ser46 of p53

Isotype

Mouse IgG1κ

Reactivity

Human p53-phosphorylated at Ser46.
Not tested in other species.

Special notes

N/AApplication

1. Western blotting (1/1,000~1/2,000)
2. Immunohistochemistry, Paraffin-embedded (assay dependent)
3. ELISA

Background

p53 mutants are found in more than half of human cancers and are consideredas the most important human cancer related gene. p53 is detected at 53kD
position by electrophoresis and is composed of 393 amino acids.

In theunstressed normal cells the p53 level is low and it is inactive. However, withstress, especially with DNA damage, it is activated to promote arrest of cell cycle
and repair of DNA damage, or induction of apoptosis. The functions of p53 areregulated by phosphorylation of serine and threonine, and acetylation of lysine
at various sites in the molecule. Among the phosphorylation sites, Ser46 isphosphorylated when DNA damage is so severe as to become unrepairable,
which leads to apoptosis by activating transcription of proapoptotic genes suchas p53AIPI.As to the kinase of phosphorylation of Ser46, involvement of DYRK2and ATM have been implicated.

Data Link

UniProtKB/Swiss-Prot P04637 (P53_HUMAN)

Please note: All products are FOR RESEARCH USE ONLY. NOT FOR USE IN DIAGNOSTIC
PROCEDURES. NOT FOR MILITARY USE.
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Data Images: 71-115 Anti-p53 p-Ser46 (human) antibody, mouse monoclonal (#36)

Fig.1. Identification of Ser46-phosphorylated p53 protein by western blotting.
Samples: Crude cell extracts of MOLT-4 untreated (left lanes) and treated with adriamycin for 24 h(right lanes).
The left panel is the result with our product and the right panel is the one obtained with the productof our competitor.
The lower panel is the whole p53 protein identified by omnipotent anti-p53 antibody.

Fig.2. Kinetics of phosphorylation of p53 at Ser46 after X-ray irradiation .
Samples of U2OS cells (human osteosarcoma) were taken at the indicated times after X-rayirradiation at 10 Gy and analyzed by western blotting with anti-p53 p-S46 antibody (#36) and antip53 antibody (non BioAcademia). Primary antibodies were diluted with “ Can Get Signal “ signalenhancer (Toyobo, Osaka).
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Fig.3. Kinetics of phosphorylation of p53 at Ser46 after UV-irradiation.
Samples of MCF7 cells (human beast cancer cell line) were taken at the indicated times after UVirradiation at 20 J/m2 and analyzed by western blotting with anti-p53 p-S46 antibody (#36) and antip53 antibody (non BioAcademia).Primary antibodies were diluted with “ Can Get Signal “ signal
enhancer (Toyobo, Osaka).

Fig.4. Immunohistochemistry of stomach cancer.
Formalin/PFU fixed paraffin-embedded section

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PROTOCOL: WESTERN BLOTTING
1. Grow the appropriate human cells such as MCF7 (breast cancer cell line) or Molt4 (leukemia cellline) to log phase and treat the cells with DNA damaging reagents such as adriamycin or UV light(see Ref 2). The cells are incubated for 16~24 h for inducing phosphorylation of p53. The untreatedcells are used as control.
2. The cells are harvested by centrifugation and lysed by adding and incubating with lysis buffer (50mM Tris-HCl pH 7.5, 150 mM NaCl, 0.5% NP-40, 50 mM NaF, 1 mM Na3VO4, 1 mM DTT, 0.1 mg/ml
E64, 0.1 mg/ml leupeptin, 0.1 mg/ml SBTI) on ice for 30 min. The supernatant is obtained bycentrifugation in microcentrifuge at 10,000 rpm for 10 min.
3. The lysates are added with SDS-sample buffer and separated by 10% SDS PAGE followed bytransfer of proteins to Immobilon P-membrane (Millipore).
4. Immunoblotting is carried out using monoclonal antibody specific for p53-phospho-Ser46 (clone#36) at 0.5~1.0μg/ml followed by horseradish peroxidase-conjugated secondary antibodies to mouse
IgG and detection with enhanced chemiluminescence (ECL-plus, GE Healthcare Biosciences). For
blocking purpose, bovine serum albumin is generally recommended for detection of phosphorylated
proteins, rather than skim milk powder.
References

This product was used in the following publications.

1. Taira N. et al (2007) DYRK2 is targeted to the nucleus and controls p53 via Ser46 phosphorylationin the apoptotic response to DNA damage. Mol. Cell 25:725-738. PMID: 17349958 WB
2. Kodama M. et al (2010) Requirement of ATM for rapid p53 phosphorylation at Ser46 without
Ser/Thr-Gln sequences. Mol Cell Biol. 30:1620-33. PMID20123963. WB
3. Taira N. et al (2010) ATM augments nuclear stabilization of DYRK2 by inhibiting MDM2 in theapoptotic response to DNA damage. J Biol Chem. 285:4909-19. PMID: 19965871 WB
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