Phospho-TP53 (Ser33) Antibody

What is the significance of p53 phosphorylation at serine 33 in cancer biology?

Phosphorylation of p53 at serine 33 is a crucial post-translational modification that occurs in response to DNA damage and cellular stress. This modification plays a significant role in p53 activation and stability. In cancer biology, phosphorylated p53 at Ser33 has been specifically observed in various malignancies, particularly in head and neck squamous cell carcinoma (HNSCC). Research has shown that in oropharyngeal squamous cell carcinoma (OPSCC), phospho-p53 S33 was expressed in 13/24 cases (54%), indicating its potential importance in cancer development and progression . This phosphorylation can modulate p53's tumor suppressor functions, including cell cycle arrest and apoptosis induction. Importantly, phosphorylation at this site appears to be more immunogenic than phosphorylation at some other p53 sites, making it a potential target for cancer immunotherapy approaches .

How do I select the appropriate Phospho-TP53 (Ser33) antibody for my experimental technique?

Selection of the appropriate Phospho-TP53 (Ser33) antibody depends on several factors including your experimental technique, species reactivity requirements, and specificity needs:

For Western Blotting:

• Choose antibodies validated specifically for WB applications with recommended dilutions typically around 1:1000

• Consider antibodies that recognize denatured epitopes and have minimal cross-reactivity

For Immunohistochemistry:

• Select antibodies specifically validated for IHC with appropriate dilutions (typically 1:50-1:250)

• For FFPE samples, ensure the antibody is validated for formalin-fixed tissues

• Consider clone-specific performance (e.g., clone 3E7 has been validated for IHC)

For Immunoprecipitation:

• Choose antibodies with high specificity and validated for IP applications

• Consider using rabbit polyclonal antibodies which often perform well in IP

For species reactivity:

• Verify the species cross-reactivity in product documentation

• Note that some antibodies have confirmed reactivity to human and monkey p53 , while others may recognize mouse and rat p53 as well

For phospho-specificity validation:

• Request documentation showing the antibody specifically recognizes the Ser33 phosphorylated form but not the unphosphorylated form

• Consider antibodies raised against synthetic phosphorylated peptides around S33 of human p53

What controls should I include when using Phospho-TP53 (Ser33) antibody in research?

When working with Phospho-TP53 (Ser33) antibody, proper controls are essential for result validation:

Positive Controls:

• Cell lines with DNA damage-induced p53 phosphorylation (e.g., UV or IR-treated CEM cells show rapid phosphorylation at Ser33)

• Cells treated with phosphatase inhibitors to preserve phosphorylation status

• Recombinant phosphorylated p53 protein standards

Negative Controls:

• p53-null cell lines (to confirm specificity)

• Non-phosphorylated p53 peptides for blocking experiments

• Samples treated with lambda phosphatase to remove phosphorylation

• Cells with CRISPR/Cas9-mediated p53 knockout

Specificity Controls:

• Parallel analysis with antibodies recognizing total p53 to normalize phospho-signal

• Comparison with antibodies recognizing other p53 phosphorylation sites (e.g., Ser15, Ser37)

• Competition assays with phosphorylated versus non-phosphorylated peptides

• Use of p53 mutants where Ser33 is substituted with alanine (S33A)

Technical Controls:

• Include isotype control antibodies matched to your primary antibody (e.g., rabbit IgG for rabbit-derived phospho-p53 antibodies)

• Secondary antibody-only controls to assess non-specific binding

• For arrays or high-throughput applications, include beta-actin and GAPDH as loading controls

How can I optimize detection of p53 Ser33 phosphorylation following DNA damage?

Optimizing detection of p53 Ser33 phosphorylation following DNA damage requires careful attention to several methodological factors:

Timing of Analysis:

• Phosphorylation at Ser33 occurs rapidly after DNA damage, with significant induction observed within 10 minutes of IR or UV treatment in some cell lines

• Consider performing a time-course analysis (10 min, 30 min, 1h, 2h, 4h, 8h) to capture the peak phosphorylation window for your specific damage model

DNA Damage Induction Methods:

• Ionizing radiation (IR): Generally produces robust and rapid phosphorylation at Ser33

• UV radiation: Also induces Ser33 phosphorylation but may follow different kinetics than IR in some cell lines

• Chemical agents: Consider etoposide, doxorubicin, or cisplatin which can upregulate phosphorylated p53

Sample Preparation:

• Immediately lyse cells in ice-cold buffer containing phosphatase inhibitors to preserve phosphorylation status

• For Western blotting, avoid repeated freeze/thaw cycles of lysates

• For IHC, optimize fixation time to prevent epitope masking while preserving tissue morphology

Detection Enhancement:

Cell Line Selection:

• CEM cells (human acute lymphoblastic leukemia) express high levels of p53 and show clear induction of Ser33 phosphorylation after DNA damage

• HT29 cells also demonstrate rapid induction of phosphorylation

• Consider using paired cell lines with wild-type and mutant p53 to compare phosphorylation patterns

What are the methodological challenges in studying p53 Ser33 phosphorylation in tumor samples?

Studying p53 Ser33 phosphorylation in tumor samples presents several methodological challenges that researchers should address:

Tissue Preservation Challenges:

• Phosphorylation marks are labile and can be lost during routine tissue processing

• Rapid fixation of tissue samples is critical (ideally within 15-30 minutes of collection)

• Standardize fixation protocols to ensure consistent phospho-epitope preservation

Tumor Heterogeneity Issues:

• Heterogeneous phosphorylation patterns may exist within different regions of the same tumor

• Consider techniques like tissue microarrays or multiple sampling to address heterogeneity

• Laser capture microdissection may help isolate specific tumor regions for analysis

Interpretative Challenges:

• Distinguishing specific phospho-p53 Ser33 signals from background staining in IHC

• Quantification methods should be standardized (H-score, percentage positive cells, or intensity scoring)

• In HNSCC samples, studies found no correlation between phosphorylated p53 expression and other parameters such as tumor stage, p16 expression, or HLA-DR expression

Technical Considerations:

• Use specific phospho-TP53 (Ser33) antibodies validated for FFPE tissues

• Include positive controls (e.g., tumors known to express phospho-p53) and negative controls

• Consider dual staining with total p53 antibodies to determine the proportion of phosphorylated to total p53

Clinical Sample Findings:

• In oropharyngeal squamous cell carcinoma, phospho-p53 S33 was expressed in 54% of cases (13/24)

• 42% of OPSCC cases showed double positivity for phospho-p53 S33 and phospho-p53 S37

• These findings provide a rational basis for targeting phosphorylated p53 proteins in immunotherapy approaches for HNSCC

How can phospho-specific arrays be used to study p53 signaling networks?

Phospho-specific arrays offer powerful approaches for analyzing p53 signaling networks in a high-throughput manner:

Array Selection and Design:

• p53 Signaling Phospho Antibody Arrays contain site-specific and phospho-specific antibodies that can profile phosphorylation events across the p53 pathway

• These arrays typically feature 196 antibodies with 6 replicates per antibody for statistical robustness

• Consider arrays that include both phosphorylated and total protein antibodies to calculate phosphorylation ratios

Sample Preparation Protocol:

• Extract proteins with non-denaturing lysis buffers to preserve native protein conformations

• Biotinylate protein samples according to manufacturer protocols

• Incubate labeled samples with the antibody array under standardized conditions

• Detect signals using dye-conjugated streptavidin and compatible fluorescent scanners

Experimental Design Considerations:

• Compare untreated vs. treated samples to identify dynamic phosphorylation changes

• Include time-course analyses to capture transient phosphorylation events

• Consider using phosphatase inhibitors in one set of samples to identify maximum phosphorylation potential

Data Analysis Approaches:

• Normalize signals to housekeeping proteins like beta-actin or GAPDH

• Calculate fold-changes in phosphorylation between experimental conditions

• Use hierarchical clustering or pathway analysis to identify coordinated phosphorylation events

• Validate key findings with orthogonal methods like Western blotting or mass spectrometry

Research Applications:

• Arrays can identify candidate biomarkers for disease states or treatment responses

• Compare normal samples to treated or diseased samples to identify disease-specific phosphorylation signatures

• Arrays can help elucidate the broader impact of specific stressors on the p53 signaling network

How can phosphorylated p53 peptides be used to elicit tumor-reactive T helper responses?

Research has demonstrated that phosphorylated p53 peptides can be utilized to stimulate tumor-reactive T helper lymphocyte (HTL) responses, offering promising immunotherapeutic approaches:

Peptide Selection and Design:

• Peptides containing phosphorylated serine residues in p53, particularly p53 22-41/Phospho-S33 and p53 22-41/Phospho-S37 (referred to as p-p53 S33 and p-p53 S37), have shown ability to elicit antigen-specific, tumor-reactive HTL responses

• The phosphorylated peptides can bind to multiple HLA-DR molecules, expanding potential coverage across patient populations

• Evidence suggests phosphorylated Ser33 might be more immunogenic than phosphorylated Ser37, consistent with experimental findings

Experimental Methodologies:

• Peripheral blood mononuclear cells (PBMCs) can be stimulated with phosphorylated p53 peptides to establish T cell lines specific for these modified epitopes

• T cell activation can be assessed through cytokine production assays or proliferation studies

• HLA restriction analysis using mouse fibroblasts expressing single HLA-DR molecules can identify which MHC class II molecules present the phosphorylated peptides

Clinical Observations:

• Substantial T cell responses to phosphorylated p53 peptides have been observed in HNSCC patients but not in healthy donors, suggesting tumor-associated induction of these responses

• HTL responses to p-p53 S33 were higher than responses to p-p53 S37, reinforcing the potentially greater immunogenicity of the Ser33 phosphorylation site

Combination Therapy Potential:

• Chemotherapeutic agents can augment responses of CD4 T cells specific for phosphorylated p53 via upregulation of phosphorylated p53 expression in tumor cells

• This synergy between phosphorylated p53 peptide vaccines and chemotherapy represents a promising approach for HNSCC immunotherapy

What methods can detect phospho-p53 (Ser33)-specific T cell responses in cancer patients?

Detecting phospho-p53 (Ser33)-specific T cell responses in cancer patients requires specialized immunological techniques:

Short-term T Cell Culture and Stimulation:

• Perform short-term culture using peptide-stimulated PBMCs from patients

• Include both phosphorylated p53 peptides (p-p53 S33) and control peptides like tetanus toxoid (TT 830-843) as a positive control

• Measure proliferation responses through incorporation of tritiated thymidine or flow cytometry-based proliferation assays

Cytokine Production Analysis:

• Enzyme-linked immunospot (ELISpot) assays to detect IFN-γ, IL-2, or other cytokine-secreting cells after peptide stimulation

• Intracellular cytokine staining followed by flow cytometry to identify and quantify responsive T cell populations

• Multiplex cytokine assays to profile the breadth of cytokine responses to phosphorylated peptides

HLA Restriction Determination:

• Use panels of mouse fibroblasts expressing single HLA-DR molecules as antigen presenting cells

• Determine which HLA class II molecules present the phosphorylated p53 peptides to the T cells

• This helps identify which patient populations might benefit from phospho-p53-targeted immunotherapies

T Cell Recognition Assays:

• Test recognition of tumor cells expressing phosphorylated p53 before and after treatment with chemotherapy

• Assess if chemotherapy enhances recognition through upregulation of phosphorylated p53 expression

• Evaluate T cell responses using functional readouts such as cytotoxicity, cytokine production, or activation marker upregulation

Clinical Findings:

• Studies have demonstrated that substantial T cell responses to phosphorylated p53 peptides exist in HNSCC patients but not in healthy donors

• HTL responses to p-p53 S33 were found to be higher than those to p-p53 S37 in patients

• This suggests that the precursors of phosphorylated p53-reactive HTLs exist in patients with HNSCC, providing a rationale for phosphorylated p53-targeted immunotherapy

What are the key differences between monoclonal and polyclonal antibodies targeting phospho-p53 (Ser33)?

Understanding the differences between monoclonal and polyclonal antibodies targeting phospho-p53 (Ser33) is crucial for selecting the right tool for specific research applications:

Monoclonal Antibodies:

Polyclonal Antibodies:

Selection Considerations:

• For qualitative detection of phospho-p53 (Ser33) in complex samples, polyclonal antibodies may offer advantages in sensitivity

• For precise epitope mapping or quantitative applications, monoclonal antibodies provide better specificity and reproducibility

• When reproducibility across experiments is critical, monoclonal antibodies are preferred

• For techniques requiring high affinity (like IP), polyclonal antibodies may perform better

How can I validate the phospho-specificity of anti-phospho-p53 (Ser33) antibodies?

Rigorous validation of phospho-specificity is essential when working with phospho-p53 (Ser33) antibodies:

Peptide Competition Assays:

• Perform parallel Western blots or immunostaining with antibody pre-incubated with:

  • Phosphorylated p53 Ser33 peptide (should block signal)

  • Unphosphorylated p53 peptide containing the same sequence (should not block signal)

  • Phosphorylated peptides from other p53 sites (should not block signal if antibody is specific)

• Signal abolishment only with the phospho-Ser33 peptide confirms specificity

Phosphatase Treatment:

• Treat half of your sample with lambda phosphatase to remove phosphorylation

• Compare phospho-p53 (Ser33) signal between treated and untreated samples

• Specific phospho-antibodies should show diminished or absent signal in phosphatase-treated samples

Mutagenesis Approach:

• Express wild-type p53 alongside p53 with serine-to-alanine mutation at position 33 (S33A)

• Induce phosphorylation (e.g., with DNA damaging agents)

• Specific antibodies should recognize only wild-type p53 after induction, not the S33A mutant

Kinase Inhibition/Activation:

• Treat cells with kinase inhibitors known to affect p53 Ser33 phosphorylation

• Verify reduced phospho-signal after inhibitor treatment

• Conversely, activate relevant kinases and confirm increased phospho-signal

Immunogen Verification:

• Confirm the antibody was raised against a synthetic phosphorylated peptide around S33 of human p53

• Review ELISA validation data showing the antibody specifically recognizes phosphorylated but not unphosphorylated peptides

Phosphorylation Induction:

• Compare antibody reactivity in untreated cells versus cells treated with DNA-damaging agents known to induce Ser33 phosphorylation

• Specific antibodies should show increased signal in damaged cells

• This has been demonstrated in cell lines like CEM and HT29, where significant induction of phosphorylation at Ser33 was observed within 10 minutes of IR or UV treatment

What technological advances are improving phospho-specific p53 antibody development?

Several technological advances are enhancing the development and application of phospho-specific p53 antibodies:

Recombinant Antibody Technology:

• Recombinant phospho-p53 (Ser33) monoclonal antibodies produced in expression systems like HEK293F cells offer improved consistency and reduced batch variation

• These antibodies can be engineered for enhanced specificity and affinity to the phosphorylated epitope

• Clones like 3E7 and ARC1528 exemplify this recombinant approach to phospho-specific antibody production

High-Throughput Screening Platforms:

• Antibody arrays featuring multiple site-specific and phospho-specific antibodies enable simultaneous profiling of numerous phosphorylation events

• p53 Signaling Phospho Antibody Arrays containing 196 antibodies with 6 replicates per antibody allow comprehensive phosphorylation analysis

• These platforms enable researchers to study phosphorylation networks in various experimental conditions

Structural Biology Integration:

• Crystal structure information about p53 phosphorylation sites is being used to design more specific immunogens

• Structural analysis helps identify optimal peptide length and flanking sequences for generating antibodies that can distinguish between closely located phosphorylation sites

Multiplexed Detection Systems:

• Development of multiplexed detection methods allows simultaneous analysis of multiple p53 phosphorylation sites

• These approaches help understand the combinatorial effects of phosphorylation at different sites (e.g., Ser33 and Ser37)

• Research has shown that in OPSCC, 42% of cases were double positive for phospho-p53 S33 and phospho-p53 S37

Application-Specific Optimization:

• Antibodies are being optimized for specific applications with different dilution recommendations:

  • Western Blotting: 1:1000

  • Immunohistochemistry (Paraffin): 1:250

  • Flow cytometry: 1:50-1:200

  • Immunofluorescence: 1:50-1:200

• This application-specific optimization enhances performance across diverse experimental contexts

How does p53 Ser33 phosphorylation status correlate with response to therapy in cancer patients?

The relationship between p53 Ser33 phosphorylation and therapeutic response offers important insights for treatment stratification:

Chemotherapy Response Correlations:

• Chemotherapeutic agents can induce upregulation of phosphorylated p53, including at the Ser33 site

• This upregulation was confirmed in both in vitro and xenograft models

• Importantly, this enhanced phosphorylation may augment antitumor immune responses, as chemotherapy treatment enhanced the responses of CD4 T cells specific for phosphorylated p53

Immunotherapy Implications:

• The presence of phospho-p53 (Ser33)-specific T cells in cancer patients but not healthy donors suggests a tumor-associated immune response

• Patients with pre-existing T cell responses to phosphorylated p53 epitopes might be better candidates for immunotherapeutic approaches targeting these modifications

• Combined approaches using phosphorylated p53 peptides and chemotherapy could represent a promising immunotherapeutic strategy for HNSCC patients

Clinical Observations in HNSCC:

• Phosphorylated p53 expression was not found to correlate with tumor stage, p16 expression (a surrogate marker for HPV infection), or HLA-DR expression in HNSCC patients

• This suggests that phosphorylated p53 status is an independent parameter that may provide additional stratification information

• The finding that 54% of OPSCC cases expressed phospho-p53 S33 identifies a substantial subset of patients who might benefit from phospho-p53-targeted therapies

Methodological Considerations:

• Assessment of phospho-p53 (Ser33) in patient samples requires standardized IHC protocols and scoring systems

• Phospho-specific antibodies with validated specificity for the Ser33 site are essential for accurate clinical correlation studies

• Integration with other molecular markers and clinical parameters is necessary for comprehensive response prediction

What are the methodological challenges in developing phospho-p53 (Ser33) as a biomarker?

Developing phospho-p53 (Ser33) as a clinical biomarker presents several methodological challenges:

Pre-analytical Variables:

• Phosphorylation marks are notoriously labile and vulnerable to degradation during tissue collection, fixation, and storage

• Time from tissue collection to fixation significantly impacts phospho-epitope preservation

• Standardized protocols for rapid tissue preservation are essential for reliable phospho-p53 assessment

Analytical Standardization:

• Antibody selection is critical - different clones may have varying specificities and sensitivities

• Validated protocols for IHC, including antigen retrieval methods, antibody concentration, and incubation conditions, must be standardized across laboratories

• Positive and negative controls must be included in every assay run to ensure reproducibility

Quantification Challenges:

• Developing standardized scoring systems (H-score, percentage positivity, etc.) for phospho-p53 (Ser33) staining

• Addressing inter-observer variability in assessment of staining intensity

• Determining clinically relevant thresholds for "positive" status (current studies note 54% positivity in OPSCC, but the threshold for clinical significance needs validation)

Biological Interpretation:

• Understanding the biological significance of phospho-p53 (Ser33) in different cancer types and contexts

• Determining whether to assess phospho-p53 (Ser33) alone or in combination with other p53 phosphorylation sites

• Integrating phospho-p53 status with other molecular markers for comprehensive prognostic modeling

Clinical Validation Requirements:

• Large-scale, prospective studies correlating phospho-p53 (Ser33) status with clinical outcomes

• Multi-institutional standardization to ensure reproducibility of findings

• Development of companion diagnostic assays if phospho-p53 (Ser33) status is predictive of response to specific therapies

How do different post-translational modifications of p53 interact with Ser33 phosphorylation?

The interplay between Ser33 phosphorylation and other post-translational modifications of p53 creates a complex regulatory network:

Coordinated Phosphorylation Events:

• DNA damage induces phosphorylation at multiple p53 sites, including Ser15, Ser20, and Ser33

• These phosphorylation events can occur within minutes of DNA damage, suggesting coordinated signaling mechanisms

• In OPSCC, 42% of cases showed double positivity for phospho-p53 S33 and phospho-p53 S37, indicating simultaneous modification of multiple serine residues

Functional Consequences of Multiple Modifications:

• Phosphorylation at Ser15 and Ser20 reduces interaction between p53 and its negative regulator MDM2, leading to p53 stabilization

• While Ser33 phosphorylation has distinct functions, it may cooperate with these modifications to enhance p53 activation

• Tetramerization domain of p53 is required for efficient phosphorylation at Ser15, Ser20, and Ser33, suggesting structural requirements for these modifications

Kinase Networks:

• Different kinases target specific p53 phosphorylation sites:

  • ATM, ATR, and DNA-PK phosphorylate Ser15 and Ser37

  • Chk1 and Chk2 phosphorylate Ser20

  • CAK can phosphorylate Ser33 in vitro, although the kinase responsible for in vivo phosphorylation remains to be definitively established

• These kinase networks respond to different cellular stresses, creating context-specific modification patterns

Cross-talk with Other Modifications:

• Acetylation of p53 is another important modification that interacts with phosphorylation

• Recent research showed that acetylated p53 proteins can serve as targets of anti-tumor immunity

• Both hyperacetylation and hyperphosphorylation have been observed in malignancies, suggesting potential coordinative functions

Technological Approaches:

• Phospho-specific arrays enable simultaneous analysis of multiple p53 modifications

• Mass spectrometry-based approaches can identify and quantify combinations of modifications on single p53 molecules

• These technologies help elucidate how Ser33 phosphorylation functions within the broader context of p53 post-translational modifications