Phospho-TP53 (S33) Recombinant Monoclonal Antibody

What is Phospho-TP53 (S33) Recombinant Monoclonal Antibody and what epitope does it specifically recognize?

Phospho-TP53 (S33) Recombinant Monoclonal Antibody is a recombinant antibody specifically designed to recognize the human tumor protein p53 (TP53) when it is phosphorylated at the serine 33 position. The antibody is produced by extracting antibody genes from rabbits immunized with a synthesized peptide derived from the human TP53 protein phosphorylated at S33. These genes are then introduced into expression vectors, transfected into host suspension cells, and the antibodies are produced and purified using affinity chromatography techniques .

The specificity of the antibody is validated through multiple testing methods including ELISA, immunohistochemistry (IHC), and immunofluorescence (IF) to confirm its ability to specifically interact with human TP53 protein phosphorylated at the S33 site .

What are the recommended applications and optimal dilutions for Phospho-TP53 (S33) Recombinant Monoclonal Antibody?

Based on manufacturer recommendations and published protocols, the Phospho-TP53 (S33) Recombinant Monoclonal Antibody can be used in multiple applications with the following suggested dilutions:

These dilutions should be optimized for specific experimental conditions, including cell/tissue type, fixation method, and detection system .

What positive and negative controls should be used with Phospho-TP53 (S33) antibodies?

Positive controls:

• UV-irradiated cells (25 J/m²), harvested 4-8 hours post-treatment, as UV exposure induces significant phosphorylation at S33

• Cells treated with p38 MAPK activators (e.g., anisomycin)

• Cell lines with known high levels of p53 S33 phosphorylation

Negative controls:

• Cell lines with p53 S33A mutations or p53-null cells (e.g., H1299)

• Samples treated with lambda phosphatase to remove phosphorylation

• Blocking peptide competition assays to confirm antibody specificity

• p38 kinase inhibitor-treated cells, as p38 inhibition decreases phosphorylation at Ser33

For validating specificity in Western blotting, it's recommended to run phosphorylated and non-phosphorylated peptide controls alongside your experimental samples.

How is phosphorylation at S33 regulated in the p53 protein and what kinases are responsible?

Phosphorylation of p53 at S33 is primarily regulated by p38 mitogen-activated protein kinase (p38 MAPK). Research has demonstrated that p38 kinase can directly phosphorylate p53 at Ser33 and Ser46 both in vitro and in vivo . The regulation of this phosphorylation is complex and context-dependent:

• DNA damage response: UV radiation significantly increases p38 kinase activity, leading to enhanced phosphorylation of p53 at Ser33

• Signaling pathways: Components of the Ras signaling pathway contribute to activation of p38 kinase, which subsequently phosphorylates p53 at Ser33

• Protein-protein interactions: p53 and p38 kinase exist in the same physical complex, facilitating efficient phosphorylation

• Kinase specificity: While p38 directly phosphorylates Ser33 and Ser46, it indirectly influences phosphorylation at other sites including Ser15 and Ser37 through a coordinated phosphorylation cascade

Experimental evidence from cell culture studies shows that inhibition of p38 activation after UV irradiation significantly decreases phosphorylation at Ser33, confirming the crucial role of this kinase in regulating this specific modification .

What is the functional significance of p53 phosphorylation at Ser33 in cellular stress responses?

Phosphorylation of p53 at Ser33 plays critical roles in coordinating DNA repair, cell cycle regulation, and cell fate decisions in response to stress and damage . Key functional consequences include:

• Apoptotic regulation: Mutation studies replacing Ser33 with alanine (S33A) show decreased p53-mediated and UV-induced apoptosis, indicating its importance in programmed cell death pathways

• Coordinated phosphorylation: Ser33 phosphorylation appears to be a prerequisite for proper phosphorylation at other sites. Mutation of Ser33 completely blocks UV-induced phosphorylation at Ser37 and significantly decreases Ser15 phosphorylation, suggesting it functions as part of an integrated regulatory network

• Transcriptional activation: While single S33A mutation shows minimal effect on p53-dependent transcription, the double mutant S33,46A exhibits significantly reduced transactivation potential, suggesting a synergistic role with other phosphorylation sites

• Protein-protein interactions: Phosphorylation at Ser33 contributes to the binding energy for interactions between the p53 transactivation domain (TAD) and CREB-binding protein (CBP) domains, enhancing transcriptional coactivator recruitment

• Immune system activation: Phosphorylated p53 peptides containing phospho-S33 can bind to multiple HLA-DR molecules and induce T helper responses against tumor cells expressing the phosphorylated p53 protein

Research using phospho-specific antibodies has demonstrated that under normal conditions, there is a basal level of Ser33 phosphorylation that increases modestly following UV irradiation, suggesting its involvement in both normal cellular functions and stress responses .

How does phosphorylation at Ser33 interact with other post-translational modifications of p53?

Phosphorylation at Ser33 participates in a complex network of post-translational modifications (PTMs) that collectively regulate p53 function:

• Interdependence with other phosphorylation sites: Studies using site-specific mutants reveal that Ser33 phosphorylation is required for efficient phosphorylation at Ser37, which is completely abrogated when Ser33 is mutated to alanine. Additionally, the S33,46A double mutant shows dramatically reduced phosphorylation at Ser15

• Additive contributions to binding energy: Research demonstrates that successive phosphorylation events, including at Ser33, contribute in an additive manner to the free energy for binding of the p53 transactivation domain (TAD) to the CREB-binding protein (CBP) domains

• Phosphorylation cascade model: Evidence supports a model where p38 kinase-mediated phosphorylation at Ser33 initiates a phosphorylation cascade that includes Ser37 and Ser15, sites that can be phosphorylated by DNA-PK, ATM and ATR. This suggests a coordinated regulation of N-terminal phosphorylation centered on the phosphorylation at Ser33 and Ser46 by p38 kinase

• Multi-kinase complexes: The interdependence of these modifications supports the hypothesis that a coordinated phosphorylation of p53 N-terminal sites may result from an association with a complex containing several kinases, rather than independent modification events

The complete understanding of these interactions requires specialized techniques such as mass spectrometry-based PTM mapping and combination of phospho-specific antibodies to detect multiple modifications simultaneously.

What methodologies can be used to experimentally manipulate and study S33 phosphorylation in p53?

Several experimental approaches can be employed to specifically study S33 phosphorylation:

• Site-directed mutagenesis: Creating S33A (phospho-deficient) or S33D/S33E (phospho-mimetic) mutants allows investigation of the functional consequences of phosphorylation at this site. These mutants can be expressed in p53-null cells to study specific effects

• Kinase manipulation:

  • Activation of p38 MAPK using anisomycin or UV radiation to enhance S33 phosphorylation

  • Inhibition of p38 using specific inhibitors (e.g., SB203580) to prevent S33 phosphorylation

  • siRNA knockdown or CRISPR knockout of p38 MAPK to eliminate the primary kinase responsible for this modification

• Phosphopeptide generation:

  • Synthesize phosphorylated peptides chemically for in vitro binding studies

  • Enzymatic phosphorylation using p38α MAPK for biosynthetic preparation of phosphorylated p53 TAD

• Detection methods:

  • Phospho-specific antibodies for Western blotting, IHC, IF, and flow cytometry

  • Proximity ligation assay (PLA) to detect S33 phosphorylation in situ with single-molecule resolution

  • Mass spectrometry for comprehensive mapping of phosphorylation sites

• Binding studies:

  • Fluorescence anisotropy to measure binding affinity changes caused by phosphorylation

  • Co-immunoprecipitation to assess interaction with binding partners

These methodologies can be combined to provide comprehensive insights into the mechanisms and functions of S33 phosphorylation in various cellular contexts.

How does p53 Ser33 phosphorylation status change in response to different cellular stressors?

Different cellular stressors induce distinct patterns of p53 phosphorylation at Ser33:

• UV radiation: Induces significant phosphorylation at Ser33, with peak levels typically observed 4-8 hours post-treatment. This response is primarily mediated by activation of p38 MAPK

• Ionizing radiation (IR): May induce different phosphorylation patterns compared to UV, often involving ATM and ATR kinases more prominently than p38 MAPK

• Oxidative stress: Reactive oxygen species can activate p38 MAPK, potentially leading to increased Ser33 phosphorylation

• Chemotherapeutic agents: Many DNA-damaging agents enhance the responses of CD4 T cells specific for phosphorylated p53 peptides by upregulating phosphorylated p53 expression, including at Ser33

The temporal dynamics of Ser33 phosphorylation vary depending on the stressor, with some causing rapid and transient phosphorylation while others induce more sustained modifications. These differences likely contribute to the specificity of p53-mediated responses to different types of cellular damage.

To experimentally study these changes, time-course experiments using specific stressors followed by Western blotting with phospho-specific antibodies or mass spectrometry analysis are recommended approaches.

What is the significance of Ser33 phosphorylation in tumor immunology and potential immunotherapeutic applications?

Phosphorylated p53 peptides, including those containing phospho-Ser33, have shown promising results in tumor immunology with potential applications in immunotherapy:

• T helper cell responses: The p53 22-41/Phospho-S33 peptide can induce T helper responses against tumor cells expressing the phosphorylated p53 protein. These T helper lymphocytes specifically recognize and respond to the phosphorylated epitope but not to the non-phosphorylated wild-type p53 peptide

• HLA binding and population coverage: Phosphorylated p53 peptides can bind to multiple HLA-DR molecules, providing potential coverage for a broad population of cancer patients. This makes them attractive candidates for cancer immunotherapy approaches

• Enhanced responses with chemotherapy: Chemotherapeutic agents have been shown to augment the responses of CD4 T cells specific for phosphorylated p53 by upregulating phosphorylated p53 expression, suggesting potential benefits of combination approaches

• Clinical relevance: Evaluation of clinical samples from oropharyngeal squamous cell carcinoma revealed that 54% (13/24 cases) were positive for phosphorylated p53. Importantly, lymphocytes specific for phosphorylated p53 peptide epitopes were observed in head and neck squamous cell cancer (HNSCC) patients but not in healthy donors, suggesting that precursors of phosphorylated p53-reactive helper T lymphocytes exist in cancer patients

• Immunogenicity differences: Research indicates that phosphorylated Ser33 might be more immunogenic than phosphorylated Ser37, as evidenced by stronger helper T lymphocyte responses to p-p53 S33 compared to p-p53 S37

These findings suggest that combining phosphorylated p53 peptides and chemotherapy could represent a novel immunological approach to treat certain cancers, particularly those with intact p53 that undergoes stress-induced phosphorylation.

How can Phospho-TP53 (S33) antibodies be used to stratify cancer patients for personalized treatment approaches?

Phospho-TP53 (S33) antibodies have potential applications in patient stratification and personalized cancer treatment:

• Biomarker potential: Phosphorylated p53 at Ser33 could serve as a biomarker for:

  • Tumor progression and aggressiveness

  • DNA damage response pathway integrity

  • Potential responsiveness to therapies targeting p53 pathways or p38 MAPK signaling

• Patient screening for immunotherapy: Detection of phosphorylated p53 in tumor samples could help identify patients more likely to respond to immunotherapeutic approaches targeting phospho-p53 epitopes. Research has shown that 54% of oropharyngeal squamous cell carcinoma cases were positive for phosphorylated p53

• Combination therapy guidance: Since chemotherapeutic agents can upregulate phosphorylated p53 expression, measuring Ser33 phosphorylation status before and after initial treatment could help identify optimal combination strategies

• Monitoring treatment response: Serial biopsies analyzed with phospho-specific antibodies could track changes in p53 phosphorylation status during treatment to evaluate pathway engagement and efficacy

• Resistance mechanisms: Changes in Ser33 phosphorylation patterns might indicate development of resistance to certain therapies, particularly those targeting upstream kinases like p38 MAPK

Methodologically, this requires standardized protocols for tissue preparation, antibody validation across different sample types, and correlation with clinical outcomes to establish reliable cutoffs for positivity and clinical decision-making.

What role does Ser33 phosphorylation play in the differential regulation of cell cycle arrest versus apoptosis by p53?

The phosphorylation status of p53 at Ser33, particularly in combination with other modifications, appears to influence the balance between cell cycle arrest and apoptotic responses:

• Apoptosis regulation: Mutation studies replacing both Ser33 and Ser46 with alanine (S33,46A double mutant) show significantly decreased ability to induce apoptosis compared to wild-type p53. This is evidenced by:

  • Increased colony formation (5-7 times more colonies) in cells transfected with p53-S33,46A compared to wild-type p53

  • Reduced sub-G1 DNA content (indicating apoptosis) in cells expressing the S33,46A mutant after UV exposure (16-18% versus 51% for wild-type p53)

• Threshold effect: Single mutants (S33A or S46A alone) were able to suppress colony formation to the same extent as wild-type p53, suggesting a potential threshold effect where both sites need to be unphosphorylated to significantly impair apoptotic function

• Coordinated regulation: The substantial reduction in apoptosis in the S33,46A double mutant correlates with:

  • Complete abrogation of UV-induced phosphorylation on Ser37

  • Significant decrease in Ser15 phosphorylation

  This suggests Ser33 phosphorylation influences apoptotic responses both directly and through its effects on other phosphorylation events

• Protein-protein interactions: Phosphorylation at Ser33 contributes to the binding energy for interactions between the p53 transactivation domain and transcriptional coactivators like CBP, potentially influencing the spectrum of genes activated and thus cell fate decisions

These findings suggest that phosphorylation at Ser33 plays a complex role in determining cell fate, with its importance being most evident when considered as part of the broader phosphorylation network rather than in isolation.

How can researchers troubleshoot non-specific binding or weak signal issues when using Phospho-TP53 (S33) antibodies?

When encountering problems with Phospho-TP53 (S33) antibodies, researchers can implement the following troubleshooting strategies:

For non-specific binding:

• Optimize blocking conditions:

  • Test different blocking agents (BSA, non-fat dry milk, casein, commercial blockers)

  • Increase blocking time and/or concentration

  • Include 0.1-0.3% Tween-20 in wash and antibody diluent buffers

• Adjust antibody dilution:

  • Titrate the antibody using a dilution series to determine optimal concentration

  • For Western blotting, try 1:1000-1:2000 range

  • For IHC, consider 1:50-1:200 range

• Implement peptide competition:

  • Pre-incubate antibody with phosphorylated peptide to confirm specificity

  • Compare to non-phosphorylated peptide control to verify phospho-specificity

• Use appropriate controls:

  • Include p53-null or S33A mutant samples

  • Treat half of your sample with phosphatase before detection

For weak signal issues:

• Sample preparation:

  • Add phosphatase inhibitors (sodium fluoride, sodium orthovanadate, etc.) to all buffers

  • Minimize time between sample collection and processing

  • Consider using phospho-enrichment techniques

• Enhance phosphorylation signal:

  • Treat cells with UV irradiation (25 J/m²) or other p38 MAPK activators

  • Harvest cells at optimal time points after treatment (4-8 hours for UV)

• Detection enhancement:

  • Try signal amplification systems (e.g., biotin-streptavidin, tyramide)

  • For Western blotting, increase exposure time or use more sensitive substrate

  • For IHC/IF, optimize antigen retrieval methods (test different pH buffers)

• Storage and handling:

  • Aliquot antibodies to avoid freeze-thaw cycles

  • Store at recommended temperature (-20°C)

  • Check expiration date and proper storage conditions

By systematically addressing these factors, researchers can optimize their experimental conditions to achieve specific and sensitive detection of phosphorylated p53 at Ser33.

How can Phospho-TP53 (S33) antibodies be utilized in proximity ligation assays for single-molecule detection?

Proximity Ligation Assay (PLA) offers a powerful approach for detecting phosphorylated p53 at Ser33 with single-molecule resolution in situ. This technique is particularly valuable for studying low-abundance modifications in their native cellular context:

• Principle of the assay:
  The PLA requires two antibodies that bind in close proximity (< 40 nm). For phospho-TP53 (S33) detection, researchers use:

  • A rabbit polyclonal antibody recognizing the phospho-S33 epitope

  • A mouse monoclonal antibody targeting total p53

  When these antibodies bind to the same p53 molecule, oligonucleotide-conjugated secondary antibodies enable amplification and detection of a fluorescent signal, visualized as discrete spots where phosphorylated p53 is present

• Implementation protocol:

  • Fix cells using paraformaldehyde (typically 4%) and permeabilize with Triton X-100

  • Block non-specific binding sites with appropriate blocking solution

  • Incubate with primary antibodies (rabbit anti-phospho-TP53 S33 at 1:1200 dilution and mouse anti-TP53 at 1:50 dilution)

  • Add PLA probes (secondary antibodies with attached oligonucleotides)

  • Perform ligation and amplification steps according to manufacturer's protocol

  • Counterstain nuclei with DAPI and image using fluorescence microscopy

• Analysis and quantification:

  • Each red dot in the resulting images represents a single phosphorylated p53 molecule

  • Images can be analyzed using specialized software such as BlobFinder (available from The Centre for Image Analysis at Uppsala University)

  • Quantification of dots per cell provides a measure of phosphorylation levels

• Advantages over traditional methods:

  • Single-molecule sensitivity

  • Visualization of spatial distribution within cells

  • Elimination of non-specific background through requirement for dual antibody binding

  • Quantitative assessment of phosphorylation events in situ

This approach is particularly valuable for studying the dynamics of p53 phosphorylation in response to different stressors, in different subcellular compartments, or in heterogeneous cell populations within tissue samples.

How can phosphorylated p53 epitopes including S33 be utilized for cancer immunotherapy development?

The use of phosphorylated p53 epitopes for cancer immunotherapy represents an emerging approach with several promising aspects:

• Generation of phospho-epitope specific T cells:

  • Synthetic phosphorylated p53 peptides, including p53 22-41/Phospho-S33, can be used to stimulate and expand T helper cells that specifically recognize the phosphorylated epitope

  • These T cells do not respond to non-phosphorylated wild-type p53 peptides, providing tumor specificity

• Synergy with conventional cancer treatments:

  • Chemotherapeutic agents upregulate phosphorylated p53 expression in tumor cells, making them more recognizable by phospho-epitope specific T cells

  • This suggests potential benefits from combination approaches where chemotherapy sensitizes tumors to immunotherapy targeting phosphorylated p53

• Experimental protocols for T cell generation:

  • Isolate peripheral blood mononuclear cells (PBMCs) from cancer patients or healthy donors

  • Stimulate with synthetic phosphorylated p53 peptides (including p53 22-41/Phospho-S33)

  • Expand T cells using appropriate cytokines (IL-2, IL-7, etc.)

  • Test specificity using peptide-pulsed APCs and measure responses via cytokine production, proliferation, or cytotoxicity assays

• Clinical potential:

  • Analysis of head and neck squamous cell carcinoma (HNSCC) patients showed pre-existing T cell responses to phosphorylated p53 peptides, indicating natural immunogenicity

  • 54% of oropharyngeal squamous cell carcinoma samples were positive for phosphorylated p53, representing a substantial patient population that might benefit from this approach

• MHC coverage and population applicability:

  • Phosphorylated p53 peptides can bind to multiple HLA-DR molecules, providing broad population coverage

  • Experimental verification of HLA binding can be performed using panels of mouse fibroblasts expressing single HLA-DR molecules as antigen presenting cells

These findings point to a novel immunotherapeutic strategy that targets tumor-specific post-translational modifications rather than tumor-specific proteins, potentially offering greater specificity with reduced off-target effects against normal tissues.

What is the relationship between p38 MAPK signaling, p53 Ser33 phosphorylation, and therapeutic response in cancer?

The interconnection between p38 MAPK signaling, p53 Ser33 phosphorylation, and therapeutic response in cancer represents an important area for translational research:

• Signaling pathway integration:

  • p38 MAPK directly phosphorylates p53 at Ser33 and Ser46, forming a critical link between cellular stress signaling and p53 activation

  • Components of the Ras signaling pathway contribute to activation of p38 kinase and subsequent p53 phosphorylation, creating cross-talk between oncogenic and tumor suppressor pathways

  • p53 and p38 kinase exist in the same physical complex, facilitating efficient signal transduction

• Impact on therapeutic responses:

  • Inhibition of p38 activation after UV irradiation decreases phosphorylation at Ser33, Ser37, and Ser15, and markedly reduces UV-induced apoptosis in a p53-dependent manner

  • This suggests that integrity of the p38-p53 phosphorylation axis may be required for optimal response to DNA-damaging therapies

  • Conversely, upregulation of this pathway might sensitize resistant tumors to conventional treatments

• Experimental evidence from treatment models:

  • Chemotherapeutic agents augment the responses of CD4 T cells specific for phosphorylated p53 by upregulating phosphorylated p53 expression

  • This has been confirmed in both in vitro and xenograft models, suggesting clinical relevance

• Potential therapeutic approaches:

  • Activation strategy: Enhancing p38 MAPK activity in tumors with wild-type p53 could increase p53 phosphorylation and promote apoptosis

  • Inhibition strategy: In contexts where p53 is mutated or has oncogenic functions, blocking p38-mediated phosphorylation might provide therapeutic benefit

  • Immunotherapeutic approach: Using phospho-S33 peptide vaccination in combination with agents that increase p38 activity and p53 phosphorylation

• Biomarker applications:

  • Phospho-S33 status could serve as a biomarker for:

    • Functional p38-p53 signaling axis

    • Potential responsiveness to DNA-damaging therapies

    • Selection of patients for combination treatments targeting this pathway

The intricate relationship between these pathways offers multiple points for therapeutic intervention and suggests that personalized approaches based on the status of p38-p53 signaling could improve treatment outcomes in various cancer types.