Phospho-TP53 (Ser15) Antibody

What is the biological significance of p53 phosphorylation at Serine 15?

Phosphorylation of p53 at Serine 15 (Ser15) is a critical post-translational modification that regulates p53 activity in response to cellular stress. This phosphorylation event significantly influences p53's tumor suppressor functions through multiple mechanisms:

• It disrupts the interaction between p53 and its negative regulator MDM2, preventing ubiquitination and proteasomal degradation of p53

• It enhances p53-dependent transcription from responsive promoters including p21, BAX, and MDM2

• It is required for the recruitment of p53 to promoters post-stimulus

• It promotes local histone acetylation and chromatin relaxation, facilitating transcriptional activation

Studies have demonstrated that this phosphorylation occurs both after DNA damage and at basal levels in unstimulated cells, indicating its importance in both stress-induced and physiological p53 function .

Which experimental methods are most effective for detecting phosphorylated p53 at Ser15?

Several methodologies are available for detecting Phospho-p53 (Ser15), each with distinct advantages depending on your research questions:

For experiments requiring high sensitivity, HTRF and MSD-based electrochemiluminescence assays provide excellent signal-to-noise ratios for detecting subtle changes in phosphorylation levels .

How can I validate the specificity of a Phospho-p53 (Ser15) antibody?

Validating antibody specificity is crucial for reliable experimental outcomes. A comprehensive validation approach should include:

• Positive and negative controls: Use UV-irradiated cells (40 mJ/cm²) as positive controls, which show significant increase in p53-Ser15 phosphorylation within 1 hour post-treatment . Untreated cells serve as negative controls.

• Phosphorylation-site mutants: Test antibody reactivity against p53 S15A (alanine substitution) and S15D (phospho-mimic) mutants. A specific antibody will not recognize S15A but may recognize S15D .

• Phosphatase treatment: Treat positive control samples with lambda phosphatase; this should eliminate signal from a phospho-specific antibody.

• Western blot analysis: Verify a single band at the expected molecular weight (53 kDa) and compare with total p53 antibody blotting .

• Immunofluorescence:

  • In untreated cells: Look for "patchy" localization throughout nucleoplasm

  • In DNA-damaged cells: Observe closely packed foci of intense immunofluorescence

  • In mitotic cells: Check for cytoplasmic localization

For comprehensive validation, compare results across multiple detection methods to confirm consistent patterns of phosphorylation in response to stimuli .

How should I optimize Chromatin Immunoprecipitation (ChIP) protocols when using Phospho-p53 (Ser15) antibodies?

Optimizing ChIP protocols for Phospho-p53 (Ser15) requires careful consideration of multiple parameters:

• Antibody concentration: For optimal ChIP results, use 5 μL of antibody and 10 μg of chromatin (approximately 4 × 10^6 cells) per immunoprecipitation . This ratio ensures sufficient antibody for complete capture without excess that could increase background.

• Crosslinking conditions: Since phosphorylated p53 may have different DNA binding dynamics than total p53, optimize formaldehyde crosslinking time (typically 10-15 minutes at room temperature) to capture transient interactions.

• Sonication parameters: Phospho-p53 (Ser15) has been shown to influence local chromatin structure , so optimize sonication conditions to achieve chromatin fragments of 200-500 bp while preserving epitope integrity.

• Washing stringency: Balance between removing non-specific binding while maintaining specific interactions; phospho-epitopes may be more sensitive to high-salt conditions.

• Elution and reversal: Use enzymatic chromatin IP kits which have been validated with this antibody for optimal results.

• Controls: Include:

  • Input chromatin (non-immunoprecipitated)

  • IgG negative control

  • Total p53 antibody as positive control

  • ChIP for known p53 target genes (p21/CDKN1A, MDM2, BAX)

After ChIP, qPCR primer design should target regions containing p53 response elements in promoters of interest, with special attention to p21/CDKN1A which shows strong dependency on Ser15 phosphorylation .

What are the critical considerations when quantifying changes in p53-Ser15 phosphorylation relative to total p53 levels?

Accurate quantification of p53-Ser15 phosphorylation relative to total p53 requires careful methodological consideration:

• Signal normalization strategy: Calculate the ratio of phosphorylated p53 signal increase (Δp53-Ser15P) to total p53 increase (Δp53) . This approach distinguishes between:

  • Simple accumulation of total p53 (where the phosphorylation proportion remains unchanged)

  • Actual increases in the phosphorylation stoichiometry

• Cell cycle considerations: Phosphorylation levels can vary significantly by cell cycle phase:

  • After topoisomerase I inhibitor treatment, S-phase cells show higher p53-Ser15P/total p53 ratios

  • After mitoxantrone treatment, G2M cells show higher phosphorylation ratios

• Time course analysis: Maximum p53-Ser15 phosphorylation typically occurs 4-6 hours after DNA damage stimulus, which coincides with peak Chk2 activation but not ATM activation and H2AX phosphorylation (which typically peak 1-2 hours post-treatment) .

• Antibody selection: Use antibody pairs validated to work together in multiplex assays that simultaneously detect phosphorylated and total p53 .

• Sample preparation: Standardize lysis conditions to ensure complete extraction of both nuclear (where most phosphorylated p53 resides) and cytoplasmic p53 pools.

A well-designed experiment should include western blot validation alongside quantitative assays to confirm that changes in signaling are not artifacts of detection method .

How can I design experiments to distinguish between ATM-dependent and Chk2-dependent phosphorylation of p53 at Ser15?

Distinguishing between ATM-dependent and Chk2-dependent phosphorylation of p53 at Ser15 requires a multi-faceted experimental approach:

• Kinetic analysis: Based on temporal patterns, design time-course experiments:

  • ATM activation and H2AX phosphorylation typically peak 1-2 hours after DNA damage

  • Chk2 activation and maximum p53-Ser15 phosphorylation occur 4-6 hours post-treatment

• Specific inhibitors:

  • KU-55933 (ATM inhibitor): Blocks early phosphorylation events

  • Chk2 Inhibitor II: Targets later phosphorylation events

  • Compare the inhibition patterns to determine the relative contribution of each kinase

• Genetic approaches:

  • siRNA/shRNA knockdown of ATM or Chk2

  • CRISPR/Cas9-mediated knockout cells

  • Complementation with kinase-dead mutants

• Cellular localization studies:

  • ATM typically phosphorylates p53 in response to double-strand breaks

  • Chk2 shows distinct centriole localization during mitosis, which correlates with high p53-Ser15 phosphorylation levels

• Stimulus-specific analysis:

  • Topoisomerase I inhibitors (e.g., TPT) cause S-phase-specific phosphorylation

  • Compare with non-DNA damage stimuli like Nutlin-3a (MDM2 inhibitor), which induces p53 stabilization without significant DNA damage

A comprehensive phospho-proteomic approach using mass spectrometry can further distinguish the exact contribution of each kinase by identifying co-occurring phosphorylation events specific to each pathway .

What control samples should be included when measuring basal versus induced p53-Ser15 phosphorylation?

A robust experimental design for measuring p53-Ser15 phosphorylation should include these essential controls:

• Positive controls:

  • UV-irradiated cells (40 mJ/cm²): These exhibit strong p53-Ser15 phosphorylation within 1 hour of treatment

  • Topoisomerase inhibitor-treated cells: TPT or MXT treatment for 4-6 hours induces significant phosphorylation

  • Etoposide treatment: Creates double-strand breaks that trigger ATM-dependent phosphorylation

• Negative controls:

  • Untreated cells: To establish baseline phosphorylation

  • p53-null cells (e.g., H1299): To verify antibody specificity

  • Phosphatase-treated lysates: To confirm phospho-specificity

• Specialized controls:

  • Nutlin-3a treated cells: Causes p53 accumulation with minimal increase in Ser15 phosphorylation stoichiometry

  • IPTG-inducible p53 expression systems: Allow comparison of wild-type p53 with S15A and S15D mutants at identical expression levels

  • Cell cycle synchronized populations: Important because mitotic cells show constitutively high p53-Ser15 phosphorylation

• Technical controls:

  • Total p53 quantification in parallel samples

  • Loading controls (β-actin, GAPDH)

  • Signal linearity validation using serial dilutions of positive control samples

When interpreting results, remember that basal phosphorylation levels are normally present in unstimulated cells and play physiological roles, rather than representing background or non-specific signal .

How can I resolve contradictory results between different detection methods for p53-Ser15 phosphorylation?

Resolving contradictory results between different detection methods requires systematic analysis of methodological differences:

• Epitope accessibility differences:

  • Western blotting: Denatured proteins expose all epitopes

  • Immunoprecipitation/ChIP: Conformational changes or protein-protein interactions may mask epitopes

  • ELISA/HTRF: Sandwich antibody configurations require simultaneous binding of two antibodies

  Solution: Use different antibody clones targeting the same phospho-epitope but with different binding characteristics.

• Sample preparation effects:

  • Phosphatase activity during lysis can reduce signal

  • Different lysis buffers extract different subcellular pools of p53

  • Nuclear extraction efficiency varies between methods

  Solution: Include phosphatase inhibitors in all buffers; standardize extraction protocols; compare nuclear and cytoplasmic fractions separately.

• Signal calibration issues:

  • Western blot: Semi-quantitative; depends on exposure time

  • ELISA/HTRF: Quantitative but may have different dynamic ranges

  Solution: Generate standard curves using recombinant phosphorylated and non-phosphorylated p53; include gradient of positive control samples.

• Cell heterogeneity effects:

  • Population-based assays (Western blot, ELISA): Average signal across all cells

  • Single-cell methods (Flow cytometry, immunofluorescence): Reveal subpopulations

  Solution: Compare cell cycle-synchronized populations; use flow cytometry to isolate subpopulations for further analysis .

• Temporally dynamic phosphorylation:

  • Different methods may have different processing times

  Solution: Standardize sample handling time; perform time-course experiments.

When methods consistently disagree, consider that each may be measuring different pools of phosphorylated p53 with different biological significance .

What are the most common technical challenges when using Phospho-p53 (Ser15) antibodies in fixed tissue samples?

Working with Phospho-p53 (Ser15) antibodies in fixed tissue samples presents several technical challenges that require specific optimization strategies:

• Epitope masking during fixation:

  • Formalin fixation can cross-link proteins and mask phospho-epitopes

  Solution: Optimize antigen retrieval methods; compare heat-induced (citrate buffer, pH 6.0) versus enzyme-based retrieval; test different retrieval times (10-30 minutes).

• Phosphatase activity during tissue processing:

  • Delay between tissue collection and fixation can reduce phospho-signals

  Solution: Ensure rapid fixation of tissues; include phosphatase inhibitors in buffers; consider using PAXgene or other phospho-preserving fixatives.

• Non-specific background in immunohistochemistry:

  • Endogenous peroxidase activity can cause false positives

  Solution: Include proper blocking steps (hydrogen peroxide block, protein block); optimize antibody concentration (typically start at 10-15 μg/mL) ; include appropriate controls.

• Tissue heterogeneity and interpretation challenges:

  • Variable fixation across tissue sections

  • Mixed cell populations within samples

  Solution: Use multi-staining approaches to identify specific cell types; quantify signal intensity using digital image analysis.

• Quantification limitations:

  • Chromogenic IHC has limited dynamic range for quantification

  Solution: Consider fluorescent IHC for better quantification; always include a range of control samples with known phosphorylation status in each batch.

• Validation across species:

  • Antibody may have different performance across species

  Solution: Verify species cross-reactivity; the p53-Ser15 epitope is highly conserved across human, mouse and rat, but testing is advised .

When analyzing human cancer tissue samples, remember that p53 mutations may affect antibody binding or phosphorylation patterns, so correlation with molecular data on p53 mutation status can help interpretation .

How does p53-Ser15 phosphorylation impact different cellular outcomes (cell cycle arrest versus apoptosis)?

The differential impact of p53-Ser15 phosphorylation on cellular fate decisions involves complex regulatory mechanisms:

• Promoter-specific effects:

  • Cell cycle arrest genes (p21/CDKN1A): Studies show complete dependence on Ser15 phosphorylation for activation

  • Apoptotic genes (BAX): Show reduced but not abolished activation with S15A mutation

  • MDM2: Partial dependence on Ser15 phosphorylation

  This differential requirement suggests Ser15 phosphorylation may preferentially direct p53 toward cell cycle arrest programs.

• Threshold effects:

  • Low levels of Ser15 phosphorylation (as seen with Nutlin-3a treatment) may be sufficient for cell cycle arrest

  • Higher levels (observed after severe DNA damage) may be required for apoptotic gene activation

• Temporal dynamics:

  • Early/transient Ser15 phosphorylation tends to promote cell cycle arrest

  • Sustained phosphorylation, especially when combined with other modifications, shifts the balance toward apoptosis

• Cooperation with other modifications:

  • Ser15 phosphorylation facilitates subsequent phosphorylation of Ser20 by Chk1/Chk2, which enhances p53 tetramerization and stability

  • Combined Ser15 and Ser20 phosphorylation may be required for full apoptotic activity

• Cell type-specific responses:

  • Different cell types show varying dependencies on Ser15 phosphorylation

  • Correlation with cell cycle phase: S-phase cells show higher phosphorylation ratios after topoisomerase inhibitor treatment

Experimental evidence suggests that S15D phospho-mimic mutations can rescue transcriptional activity at all p53-responsive promoters, indicating that the phosphorylation is necessary but works in concert with other modifications to determine final cellular outcomes .

What is the relationship between p53-Ser15 phosphorylation and histone modifications at p53 target genes?

The relationship between p53-Ser15 phosphorylation and histone modifications represents a critical mechanistic link in transcriptional regulation:

• Recruitment of histone acetyltransferases (HATs):

  • Ser15 phosphorylation promotes p53 interaction with HATs including p300/CBP

  • This interaction is essential for acetylation of histones H3 and H4 at p53 target gene promoters

• Chromatin relaxation mechanism:

  • ChIP analyses reveal that S15A mutant p53 binds to promoters but fails to initiate local histone acetylation

  • Ser15 phosphorylation is required for chromatin relaxation at p53-responsive promoters

  • This chromatin remodeling is necessary for recruitment of RNA polymerase II and transcriptional machinery

• Sequential modification model:

  • Ser15 phosphorylation occurs first

  • This enables p53 to recruit HATs

  • HATs acetylate both p53 itself (enhancing DNA binding) and local histones

  • The resulting open chromatin structure allows additional transcription factors to bind

• Promoter-specific effects:

  • Different p53 target genes show varying dependencies on histone modifications

  • p21/CDKN1A promoter shows stronger dependence on Ser15 phosphorylation-mediated histone acetylation than BAX or MDM2

• Cell cycle-dependent patterns:

  • Mitotic cells show distinct patterns of p53-Ser15 phosphorylation and H2AX phosphorylation

  • These patterns may reflect different chromatin states across the cell cycle

To experimentally investigate this relationship, ChIP-reChIP experiments (sequential immunoprecipitation) can be performed using anti-phospho-p53(Ser15) antibody followed by antibodies against modified histones (e.g., H3K9ac, H3K4me3) to determine their co-occurrence at specific genomic loci .

How do different DNA-damaging agents influence the kinetics and magnitude of p53-Ser15 phosphorylation?

Different DNA-damaging agents produce distinct patterns of p53-Ser15 phosphorylation through varied mechanisms:

• Topoisomerase inhibitors:

  • Topoisomerase I inhibitors (TPT/Topotecan): Induce strong S-phase-specific phosphorylation

  • Topoisomerase II inhibitors (MXT/Mitoxantrone): Cause more uniform phosphorylation across cell cycle phases

  • Both reach maximum p53-Ser15 phosphorylation 4-6 hours after treatment

• Radiation-induced damage:

  • UV irradiation (40 mJ/cm²): Rapid phosphorylation within 1 hour; primarily ATR-dependent

  • Ionizing radiation: Rapid ATM-dependent phosphorylation; can be detected within 15-30 minutes

• Chemical agents:

  • Alkylating agents: Slower phosphorylation kinetics

  • Crosslinking agents: Variable patterns depending on repair pathway activation

  • Replication inhibitors: S-phase specific phosphorylation

• Non-genotoxic p53 activators:

  • Nutlin-3a (MDM2 inhibitor): Causes p53 accumulation with minimal increase in Ser15 phosphorylation stoichiometry

  • Demonstrates that basal Ser15 phosphorylation is sufficient for function when p53 is stabilized

• Kinase pathway specificity:

  • ATM-dependent phosphorylation: Predominant after double-strand breaks

  • ATR-dependent phosphorylation: Major pathway after UV damage and replication stress

  • DNA-PK-dependent phosphorylation: Contributes following certain types of DNA damage

  • Chk1/Chk2-dependent phosphorylation: Secondary wave following ATM/ATR activation

For experimental design, consider that the ratio of phosphorylated to total p53 (Δp53-Ser15P/Δp53) provides more meaningful data than absolute phosphorylation levels, as it distinguishes between simple p53 accumulation and increased phosphorylation stoichiometry .

How can Phospho-p53 (Ser15) detection be integrated into multiplexed phospho-proteomic workflows?

Integrating Phospho-p53 (Ser15) detection into multiplexed phospho-proteomic workflows requires strategic experimental design:

• Mass spectrometry-based approaches:

  • Enrichment strategies: Use antibodies against Phospho-p53 (Ser15) for immunoprecipitation prior to MS analysis

  • Targeted MS methods: Develop Multiple Reaction Monitoring (MRM) or Parallel Reaction Monitoring (PRM) assays specific for p53-Ser15 phosphopeptides

  • Internal standards: Include synthetic phosphopeptides containing the Ser15 site as quantitative references

• Multiplexed antibody-based detection:

  • HTRF technology: Allows simultaneous detection of phospho-p53 and total p53

  • MSD platform: Electrochemiluminescence-based detection enables multiplexing of phospho-p53 with other phospho-proteins in the same pathway

  • Multiplex flow cytometry: Combine Phospho-p53 (Ser15) antibodies with markers for cell cycle, DNA damage (γH2AX), and apoptosis

• High-content imaging approaches:

  • Multiplex immunofluorescence: Combine with other phospho-epitopes (ATM, Chk2, H2AX)

  • Quantitative image analysis: Measure nuclear:cytoplasmic ratios, foci formation, and co-localization with DNA damage markers

• Single-cell applications:

  • Mass cytometry (CyTOF): Incorporate metal-tagged Phospho-p53 (Ser15) antibodies into panels with other phospho-proteins

  • Single-cell phospho-proteomics: Emerging techniques for measuring phosphorylation in individual cells

• Temporal dynamics analysis:

  • Live-cell reporters: Develop FRET-based sensors to monitor p53-Ser15 phosphorylation in real-time

  • Kinetic measurements: Establish time-resolved assays to capture phosphorylation dynamics

When designing multiplexed assays, ensure antibody compatibility (species, isotypes) and validate that detection antibodies do not compete for overlapping epitopes. Cross-validate findings with orthogonal methods to confirm specificity in complex samples .

What are the emerging applications of Phospho-p53 (Ser15) antibodies in cancer diagnostics and treatment monitoring?

Phospho-p53 (Ser15) antibodies are finding increasing utility in clinical applications:

• Predictive biomarker applications:

  • Treatment response prediction: p53-Ser15 phosphorylation levels can predict sensitivity to DNA-damaging chemotherapeutics

  • Resistance mechanisms: Defective phosphorylation pathways correlate with treatment resistance

  • Patient stratification: Different patterns of phosphorylation may identify patients likely to benefit from specific therapies

• Pharmacodynamic monitoring:

  • Early response assessment: Measure p53-Ser15 phosphorylation as an early pharmacodynamic marker of drug activity

  • Optimal timing: Establish time-dependent patterns after treatment to determine optimal assessment windows

  • Minimal residual disease: Monitor restoration of normal p53 signaling after therapy

• Liquid biopsy developments:

  • Circulating tumor cells (CTCs): Analysis of p53-Ser15 phosphorylation in CTCs as a minimally invasive biomarker

  • Extracellular vesicles: Detection of phospho-p53 in tumor-derived exosomes

  • Cell-free DNA studies: Correlation between circulating tumor DNA and phospho-p53 status in tumor tissues

• Advanced tissue diagnostics:

  • Multiplex immunohistochemistry: Combined detection of phospho-p53 with other markers

  • Digital pathology: Automated quantification of nuclear phospho-p53 staining

  • Spatial transcriptomics integration: Correlate phospho-p53 status with local gene expression patterns

• Therapeutic targeting applications:

  • Monitoring ATM/ATR inhibitor efficacy: Changes in p53-Ser15 phosphorylation as target engagement biomarker

  • Combination therapy rationale: Identify synergistic combinations based on phosphorylation patterns

  • Synthetic lethality approaches: Target cells with specific p53 phosphorylation defects

Recent studies have shown that analyzing p53-Ser15 phosphorylation patterns in breast cancer tissue can provide prognostic information beyond p53 mutation status alone , suggesting value in incorporating this marker into routine cancer diagnostics.

How can I design experiments to study the interplay between p53-Ser15 phosphorylation and other post-translational modifications?

Designing experiments to elucidate the interplay between p53-Ser15 phosphorylation and other post-translational modifications requires sophisticated methodological approaches:

• Sequential modification studies:

  • Time-course experiments: Track the order of appearance of different modifications

  • Site-directed mutagenesis: Create Ser15 phospho-mimetic (S15D) or phospho-dead (S15A) mutants and analyze effects on other modifications

  • Inducible expression systems: Control p53 expression level to normalize comparisons between wild-type and mutant proteins

• Modification-specific antibody combinations:

  • Multiplex immunoassays: Simultaneously detect multiple modifications (phosphorylation, acetylation, methylation, ubiquitination)

  • Sequential immunoprecipitation (IP-reIP): Use phospho-p53 (Ser15) antibody for first IP, followed by antibodies against other modifications

  • Proximity ligation assays: Detect co-occurrence of multiple modifications on the same p53 molecule

• Mass spectrometry-based approaches:

  • Top-down proteomics: Analyze intact p53 to preserve modification combinations

  • Middle-down strategies: Analyze large p53 fragments to maintain modification patterns

  • Targeted MS: Develop assays for specific combinations of modifications

  • Crosslinking MS: Identify interaction partners specific to phosphorylated p53

• Functional correlation experiments:

  • ChIP-seq with modification-specific antibodies: Compare genomic binding profiles

  • Promoter-specific activities: Test effects of modification combinations on different p53 target genes

  • Protein-protein interaction studies: Identify differential binding partners depending on modification status

• Mathematical modeling approaches:

  • Develop kinetic models of p53 modification networks

  • Predict and test modification dependencies and hierarchies

  • Integrate experimental data with computational approaches to understand emergent properties

To specifically study interactions between Ser15 phosphorylation and other key modifications, consider:

• Ser15 and Ser20: Both are DNA damage-responsive and may cooperate in MDM2 regulation

• Ser15 and K382 acetylation: Phosphorylation may promote acetylation through HAT recruitment

• Ser15 and ubiquitination: Phosphorylation inhibits MDM2-mediated ubiquitination