Phospho-TP53 (Ser46) Antibody
Description
Introduction to Phospho-TP53 (Ser46) Antibody
The Phospho-TP53 (Ser46) antibody is a specialized immunoglobulin designed to recognize and bind specifically to the p53 protein only when it has been phosphorylated at the serine 46 residue. p53, often described as the "guardian of the genome," functions as a master tumor suppressor that regulates cellular responses to various stressors, particularly DNA damage. The phosphorylation status of p53 at different residues, including Ser46, critically determines its functional outcomes in cells .
These antibodies are available in various formats, including rabbit polyclonal, mouse monoclonal, and fluorescently conjugated versions, each offering specific advantages depending on the experimental application. The antibodies have been extensively validated for research techniques such as Western blotting, immunohistochemistry, immunofluorescence, and flow cytometry .
Role in Apoptotic Pathway Activation
Phosphorylation of p53 at Ser46 represents a critical regulatory modification that directs cellular fate toward apoptosis following DNA damage. Unlike other phosphorylation sites on p53, Ser46 phosphorylation appears to be specifically involved in activating pro-apoptotic gene transcription rather than cell cycle arrest .
Research has demonstrated that Ser46 phosphorylation regulates the ability of p53 to induce apoptosis by enabling the preferential transactivation of pro-apoptotic genes . This phosphorylation event serves as a molecular switch that helps determine whether damaged cells will undergo repair or programmed cell death.
Kinases Responsible for Ser46 Phosphorylation
Several protein kinases have been identified as responsible for phosphorylating p53 at Ser46 under various stress conditions:
The ATM kinase is particularly notable as it has been shown to be responsible for the rapid phosphorylation of p53 at Ser46 during the early-phase response to DNA damage. Intriguingly, this phosphorylation occurs despite Ser46 not being located within an SQ motif, which is the canonical consensus sequence for ATM substrates .
Downstream Effects of Ser46 Phosphorylation
The phosphorylation of p53 at Ser46 triggers several downstream molecular events:
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Interaction with Pin1 (peptidyl-prolyl cis/trans isomerase): Ser46 phosphorylation facilitates p53's interaction with Pin1, which mediates conformational changes in p53 .
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Dissociation from iASPP (inhibitor of apoptosis-stimulating protein of p53): This dissociation enables p53 to induce pro-apoptotic target genes .
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Activation of specific target genes: Ser46 phosphorylation directs p53 to activate specific pro-apoptotic genes, including palmdelphin, which has been identified as a direct target of Ser46-phosphorylated p53 .
Antibody Formats and Properties
| Property | Rabbit Polyclonal | Mouse Monoclonal | Fluorescently Conjugated |
|---|---|---|---|
| Host | Rabbit | Mouse | Mouse |
| Isotype | IgG | IgG1 | IgG1 |
| Specificity | Detects endogenous p53 only when phosphorylated at Ser46 | Specifically recognizes Ser46 phosphorylated p53 | Specifically recognizes Ser46 phosphorylated p53 |
| Form | Liquid | Liquid | Liquid |
| Purification | Antigen affinity purification | Protein G purification | Protein G purification |
| Storage Buffer | PBS with glycerol and sodium azide | PBS with glycerol and preservatives | PBS with glycerol, BSA, and preservatives |
| Observed MW | 53 kDa | 53 kDa | 53 kDa |
Reactivity and Applications
The antibodies exhibit specific reactivity patterns and are validated for various applications:
DNA Damage Response Studies
Phospho-TP53 (Ser46) antibodies have been extensively used to study the cellular response to DNA damage. Research has demonstrated that following DNA damage, p53 becomes phosphorylated at Ser46, which can be readily detected using these antibodies. This phosphorylation is critical for the activation of apoptotic pathways in irreparably damaged cells .
Cancer Research Applications
These antibodies have significant applications in cancer research, given the central role of p53 dysfunction in oncogenesis. Studies have utilized Phospho-TP53 (Ser46) antibodies to:
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Investigate the apoptotic response in cancer cells following treatment with chemotherapeutic agents
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Study the impact of p53 mutations on Ser46 phosphorylation and apoptotic potential
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Evaluate the effectiveness of experimental cancer therapies aimed at restoring p53 function
Heat Stress Response Investigation
Research has revealed that heat stress induces p53 phosphorylation at Ser46, leading to mitochondrial translocation of p53 and subsequent apoptosis in vascular endothelial cells. Phospho-TP53 (Ser46) antibodies have been instrumental in elucidating this pathway, demonstrating that heat stress causes increased p53 phosphorylation at Ser46, facilitating interactions with Pin1 and promoting mitochondria-mediated apoptosis .
Ser46 Phosphorylation in Mouse Models
Studies using knock-in mice with a Ser46 to Alanine mutation (p53hki(S46A)) have provided valuable insights into the physiological roles of Ser46 phosphorylation. These studies revealed that:
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p53 stabilization in response to DNA damage is modestly reduced in cells from p53hki(S46A) mice
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p53-dependent apoptosis is partially impaired in thymocytes and E1A/Ras-expressing MEFs from these mice
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p53hki(S46A) MEFs are more easily immortalized and escape from Ras-induced senescence more readily than wild-type MEFs
These findings confirm that Ser46 phosphorylation activates p53-dependent apoptosis induced by DNA damage and cellular senescence induced by oncogenic stress .
Identification of Novel Target Genes
Recent research has identified palmdelphin as a pro-apoptotic gene induced by p53 in a phosphorylated Ser46-specific manner. This study demonstrated that:
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Upregulation of palmdelphin was observed in wild-type p53-transfected cells but not in Ser46-mutated cells
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Expression of palmdelphin was induced by p53 in response to DNA damage
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Palmdelphin induced apoptosis when expressed
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Downregulation of palmdelphin resulted in necroptosis-like cell death via ATP depletion
These findings define palmdelphin as a direct target of Ser46-phosphorylated p53 that controls cell death in response to DNA damage .
Role in Heat Stroke Pathophysiology
A recent study revealed the importance of Ser46 phosphorylation in heat stress-induced endothelial cell apoptosis. The research demonstrated that:
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Heat stress caused an increase in p53 phosphorylation at Ser46
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This phosphorylation facilitated interactions with Pin1
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This interaction promoted the localization of p53 to mitochondria, triggering apoptosis
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Reactive oxygen species (ROS) production was identified as a critical mediator in heat stress-induced Pin1/p53 signaling
These findings contribute to our understanding of the mechanisms underlying heat stress-induced endothelial dysfunction and potential therapeutic strategies for heat stroke .
Product Specs
Target Background
- This review summarizes the diverse functions of p53 in adipocyte development and adipose tissue homeostasis. It also explores the manipulation of p53 levels in adipose tissue depots and the impact on systemic energy metabolism in the context of insulin resistance and obesity. [review] PMID: 30181511
- This study reveals that a USP15-dependent lysosomal pathway controls p53-R175H turnover in ovarian cancer cells. PMID: 29593334
- The results suggest that the underlying mechanisms by which etoposide and ellipticine regulate CYP1A1 expression differ and may not be solely linked to p53 activation. PMID: 29471073
- This study examined the association of tumor protein p53 and drug metabolizing enzyme polymorphisms with clinical outcome in patients with advanced nonsmall cell lung cancer. PMID: 28425245
- POH1 knockdown induced cell apoptosis through increased expression of p53 and Bim. PMID: 29573636
- This study identified a previously unknown effect of chronic high fat diet on beta-cells, where persistent oxidative stress leads to p53 activation and subsequent inhibition of mRNA translation. PMID: 28630491
- Diffuse large B cell lymphoma lacking CD19 or PAX5 expression were more likely to have mutant TP53. PMID: 28484276
- This study demonstrates that proliferation potential-related protein promotes esophageal cancer cell proliferation and migration, and suppresses apoptosis by mediating the expression of p53 and IL-17. PMID: 30223275
- HIV-1 infection and subsequent reverse transcription are inhibited in HCT116 p53(+/+) cells compared to HCT116 p53(-/-) cells. Tumor suppressor gene p53 expression is upregulated in non-cycling cells. The restriction of HIV by p53 is linked to the suppression of ribonucleotide reductase R2 subunit expression and phosphorylation of SAMHD1 protein. PMID: 29587790
- Studies have shown that MDM2 and MDMX are targetable vulnerabilities within TP53-wild-type T-cell lymphomas. PMID: 29789628
- A significant increase in the expression of p53 and Bax was observed in cells treated with alpha-spinasterol, while cdk4/6 were significantly down-regulated upon exposure to alpha-spinasterol. PMID: 29143969
- A significant correlation was found between telomere dysfunction indices, p53, oxidative stress indices, and malignant stages of GI cancer patients. PMID: 29730783
- PGEA-AN modulates the P53 system, leading to the death of neuroblastoma cells without affecting the renal system in vivo. This suggests its potential as an anticancer agent against neuroblastoma. PMID: 29644528
- These data indicate that activation of autophagy reduces the expression of STMN1 and p53, and the migration and invasion of cancer cells. This contributes to the anti-cancer effects of Halofuginone. These findings may provide novel insights into breast cancer prevention and therapy. PMID: 29231257
- miR-150 suppresses cigarette smoke-induced lung inflammation and airway epithelial cell apoptosis by repressing p53 expression and NF-kappaB activity. PMID: 29205062
- Tumors harboring TP53 mutations, which can impair epithelial function, exhibit a unique bacterial consortium that is more abundant in smoking-associated tumors. PMID: 30143034
- This review discusses the interplay between p53, lipid metabolism, insulin resistance, inflammation, and oxidative stress in Non-alcoholic fatty liver disease. [review] PMID: 30473026
- Ubiquitin-conjugating enzyme E2S (UBE2S) enhances the ubiquitination of p53 protein, facilitating its degradation in hepatocellular carcinoma (HCC) cells. PMID: 29928880
- p53 knockout compensates osteopenia in murine Mysm1 deficiency. PMID: 29203593
- SIRT1 plays a crucial protective role in regulating the aging and apoptosis of ADSCs induced by H2O2. PMID: 29803744
- 133p53 promotes tumor invasion via IL-6 through activation of the JAK-STAT and RhoA-ROCK pathways. PMID: 29343721
- Mutant TP53 G245C and R273H can lead to more aggressive phenotypes and enhance cancer cell malignancy. PMID: 30126368
- PD-L1, Ki-67, and p53 staining individually had significant prognostic value for patients with stage II and III colorectal cancer. PMID: 28782638
- This study of patients with ccRCC, pooled analysis, and multivariable modeling demonstrated that three recurrently mutated genes, BAP1, SETD2, and TP53, have statistically significant associations with poor clinical outcomes. Notably, mutations of TP53 and SETD2 were associated with decreased CSS and RFS, respectively. PMID: 28753773
- This study revealed that the Wnt/beta-catenin signaling pathway and its major downstream target, c-Myc, increased miR552 levels. miR552 directly targets the p53 tumor suppressor, suggesting a crucial link between functional loss of APC, leading to aberrant Wnt signals, and the absence of p53 protein in colorectal cancer. PMID: 30066856
- High levels of glucose lead to endothelial dysfunction via TAF1-mediated p53 Thr55 phosphorylation and subsequent GPX1 inactivation. PMID: 28673515
- Although tumor protein p53 (p53) does not directly control the luminal fate, its loss facilitates the acquisition of mammary stem cell (MaSC)-like properties by luminal cells, predisposing them to the development of mammary tumors with loss of luminal identity. PMID: 28194015
- Fifty-two percent of patients diagnosed with glioma/glioblastoma exhibited a positive TP53 mutation. PMID: 29454261
- The expression of Ser216pCdc25C was also increased in the combined group, indicating that irinotecan likely radiosensitized the p53-mutant HT29 and SW620 cells through the ATM/Chk/Cdc25C/Cdc2 pathway. PMID: 30085332
- In the former, p53 binds to the CDH1 (encoding E-cadherin) locus to antagonize EZH2-mediated H3K27 trimethylation (H3K27me3) to maintain high levels of acetylation of H3K27 (H3K27ac). PMID: 29371630
- Among the hits, miR-596 was identified as a regulator of p53. Overexpression of miR-596 significantly increased p53 at the protein level, inducing apoptosis. PMID: 28732184
- Apoptosis pathways are impaired in fibroblasts from patients with SSc, leading to chronic fibrosis. However, the PUMA/p53 pathway may not be involved in the dysfunction of apoptosis mechanisms in fibroblasts of patients with SSc. PMID: 28905491
- Low TP53 expression is associated with drug resistance in colorectal cancer. PMID: 30106452
- The activation of p38 in response to low doses of ultraviolet radiation was postulated to be protective for p53-inactive cells. Therefore, MCPIP1 may favor the survival of p53-defective HaCaT cells by sustaining the activation of p38. PMID: 29103983
- TP53 missense mutations are associated with castration-resistant prostate cancer. PMID: 29302046
- P53 degradation is mediated by COP1 in breast cancer. PMID: 29516369
- Combined inactivation of the XRCC4 non-homologous end-joining (NHEJ) DNA repair gene and p53 efficiently induces brain tumors with hallmark characteristics of human glioblastoma. PMID: 28094268
- This study establishes a direct link between Y14 and p53 expression, suggesting a role for Y14 in DNA damage signaling. PMID: 28361991
- TP53 Mutation is associated with Mouth Neoplasms. PMID: 30049200
- Cryo-Electron Microscopy studies on p53-bound RNA Polymerase II (Pol II) reveal that p53 structurally regulates Pol II to affect its DNA binding and elongation, providing new insights into p53-mediated transcriptional regulation. PMID: 28795863
- Increased nuclear p53 phosphorylation and PGC-1alpha protein content immediately following SIE but not CE suggests these may represent important early molecular events in the exercise-induced response to exercise. PMID: 28281651
- The E6/E7-p53-POU2F1-CTHRC1 axis promotes cervical cancer cell invasion and metastasis. PMID: 28303973
- Accumulated mutant-p53 protein suppresses the expression of SLC7A11, a component of the cystine/glutamate antiporter, system xC(-), through binding to the master antioxidant transcription factor NRF2. PMID: 28348409
- Consistently, forced expression of p53 significantly stimulated ACER2 transcription. Notably, p53-mediated autophagy and apoptosis were markedly enhanced by ACER2. Depletion of the essential autophagy gene ATG5 revealed that ACER2-induced autophagy facilitates its effect on apoptosis. PMID: 28294157
- Results indicate that LGASC of the breast is a low-grade triple-negative breast cancer that harbors a basal-like phenotype with no androgen receptor expression, and shows a high rate of PIK3CA mutations but no TP53 mutations. PMID: 29537649
- This study shows an inhibitory effect of wild-type P53 gene transfer on graft coronary artery disease in a rat model. PMID: 29425775
- Our findings suggest that TP53 c.215G>C, p. (Arg72Pro) polymorphism may be considered as a genetic marker for breast cancer predisposition in the Moroccan population. PMID: 29949804
- Higher levels of the p53 isoform, p53beta, predict better prognosis in patients with renal cell carcinoma through enhancing apoptosis in tumors. PMID: 29346503
- TP53 mutations are associated with colorectal liver metastases. PMID: 29937183
- High expression of TP53 is associated with oral epithelial dysplasia and oral squamous cell carcinoma. PMID: 29893337
Q&A
How do I validate the specificity of Phospho-TP53 (Ser46) antibodies in Western blotting?
To confirm antibody specificity:
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Use phosphorylation-deficient controls: Compare lysates from wild-type cells versus TP53-knockout cells or cells treated with serine/threonine phosphatase inhibitors (e.g., calyculin A) .
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Peptide competition assays: Pre-incubate the antibody with a phosphorylated Ser46 peptide (11–60 aa region). A ≥80% signal reduction in Western blotting confirms epitope specificity .
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Orthogonal validation: Correlate phosphorylation signals with functional outcomes (e.g., apoptosis assays in UV-treated HT-29 cells) .
| Validation Method | Expected Outcome | Key Controls |
|---|---|---|
| Knockout cells | No band at 53 kDa | Wild-type vs. TP53⁻/⁻ lysates |
| Phosphatase treatment | Reduced signal intensity | ± λ-phosphatase incubation |
| Peptide blocking | Signal abolished | 1:5 molar ratio of peptide:antibody |
What are the optimal dilution ranges for this antibody across common applications?
Dilution parameters vary by experimental system:
Critical Note: Titration curves should span ±25% of the recommended range to account for cell/tissue-specific epitope accessibility .
How does Ser46 phosphorylation status correlate with p53 functional outcomes?
Phospho-Ser46 serves as a molecular switch between cell cycle arrest and apoptosis:
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DNA damage contexts:
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Functional assays:
How to resolve contradictions in Ser46 phosphorylation kinetics across studies?
Discrepancies often arise from:
Case Example: In ATM-deficient cells, Ser46 phosphorylation after IR is abolished at 1 hr but detectable at 6 hr via p38 backup . Always couple phospho-specific staining with kinase inhibitor controls (e.g., 10 µM KU55933 for ATM).
What experimental designs robustly link Ser46 phosphorylation to downstream phenotypes?
Adopt a multi-modal approach:
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Time-resolved phosphoproteomics:
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0–24 hr post-damage sampling at 2-hr intervals
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Parallel measurement of p53 tetramerization (native PAGE) and promoter occupancy (ChIP)
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Allelic replacement models:
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Generate isogenic S46A mutants via CRISPR-HDR in TP53⁺/⁺ backgrounds
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Phenotypic endpoints:
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Apoptosis (caspase-3/7 activity)
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Senescence (SA-β-gal + EdU incorporation)
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Kinase redundancy testing:
Condition siRNA Target Expected pS46 Change IR + ATM inhibitor ATM ≥70% reduction UV + p38 inhibitor p38α/β Delayed peak (6→12 hr)
Data Interpretation: In S46A MEFs, Ras-induced senescence is attenuated (20–30% SA-β-gal⁺ cells vs. 60% in WT) , requiring compensation analysis via p21/CDKN1A knockout.
How to troubleshoot subcellular localization artifacts in immunofluorescence?
Phospho-Ser46 p53 exhibits stress-dependent compartmentalization:
| Localization | Physiological Context | Common Artifacts | Solutions |
|---|---|---|---|
| Nuclear foci | Early DNA damage response (ATM activation) | Over-fixation-induced aggregation | Limit PFA fixation to 15 min |
| Diffuse nucleoplasm | Senescence-associated secretion | Cross-reactivity with autofluorescent vesicles | Include λ-phosphatase-treated controls |
| Cytoplasmic | MDM2-mediated export during recovery | Incomplete permeabilization | Titrate Triton X-100 (0.1–0.3%) |
Quantitative Example: In etoposide-treated HT-29 cells, valid phospho-Ser46 signals show >50% co-localization with γ-H2AX foci . Exclude samples with pan-nuclear staining lacking focal structures.
What are the critical controls for ChIP-seq using Phospho-Ser46 antibodies?
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IgG control: Species-matched non-immune IgG at matching concentrations
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Input normalization: Spike-in chromatin (e.g., Drosophila S2 DNA) for cross-sample comparison
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Target validation:
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siRNA-mediated p53 knockdown → ≥80% reduction in promoter pull-down
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S46A mutant cells → loss of apoptotic gene enrichment (e.g., BAX, PUMA)
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Data Quality Metrics:
| Parameter | Acceptable Range |
|---|---|
| Peak overlap with p53 consensus motifs | ≥70% |
| Reproducibility (IDR) | <0.05 |
| Signal-to-noise (FRiP) | >1% |
How to profile Ser46 phosphorylation dynamics in live cells?
Recommended Workflow:
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FRET-based biosensors:
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Design: CFP-p53(1–92)-linker-YFP + phospho-Ser46 nanobody
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Calibration: Treat with 10 µM Nutlin-3 to stabilize p53, then 10 Gy IR
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Flow cytometry optimization:
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Fixation: BD Cytofix/Cytoperm™ (20 min, 4°C)
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Co-staining: Anti-pS46 (1:100) + anti-pS15 (1:500) for multisite analysis
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Data normalization:
