Phospho-TP63 (Ser395) Antibody

What is Phospho-TP63 (Ser395) Antibody and what does it detect?

Phospho-TP63 (Ser395) antibody is a specialized immunological reagent designed to recognize the tumor protein 63 (p63) only when phosphorylated at serine 395. This phosphorylation-specific antibody detects endogenous levels of p63 protein exclusively in its phosphorylated state at the S395 position . The antibody is generated using synthesized phospho-peptides derived from human p63 around the phosphorylation site, typically including amino acids 361-410 .

P63 belongs to the p53 family of transcription factors and contains several functional domains including an N-terminal transactivation domain, a central DNA-binding domain, and an oligomerization domain . Through alternative splicing and the use of different promoters, p63 exists in multiple isoforms with varying functional properties . These isoforms play critical roles in diverse biological processes including skin development and maintenance, adult stem/progenitor cell regulation, and other tissue-specific functions .

What are the primary applications for Phospho-TP63 (Ser395) Antibody?

Phospho-TP63 (Ser395) antibody is primarily utilized in:

• Western Blot (WB): The antibody can effectively detect phosphorylated p63 protein in cell or tissue lysates with recommended dilution ranges of 1:500 to 1:2000 . Western blot analysis allows researchers to determine the relative abundance and molecular weight of phosphorylated p63.

• Enzyme-Linked Immunosorbent Assay (ELISA): The antibody performs well in ELISA applications with a recommended dilution of 1:40000 . Both standard ELISA and cell-based ELISA formats can be utilized.

• Cell-Based ELISA: Specialized colorimetric cell-based ELISA kits are available that use this antibody to detect phospho-p63 expression directly in cultured cells, enabling high-throughput screening of compounds that may affect p63 phosphorylation status . These assays can detect phospho-p63 expression in as few as 5,000 cells .

What species reactivity does Phospho-TP63 (Ser395) Antibody demonstrate?

The commercially available Phospho-TP63 (Ser395) antibodies show cross-reactivity with:

• Human p63 phosphorylated at Ser395

• Mouse p63 phosphorylated at Ser395

• Rat p63 phosphorylated at Ser395

This multi-species reactivity makes these antibodies versatile tools for comparative studies across different model organisms. The conservation of this phosphorylation site across species suggests its functional importance in p63 regulation.

How should Phospho-TP63 (Ser395) Antibody be stored and handled for optimal results?

For maximum stability and performance, researchers should follow these storage and handling guidelines:

• Storage Temperature: Store the antibody at -20°C for up to 1 year from the date of receipt .

• Formulation: The antibody is typically supplied in liquid form in PBS containing 50% glycerol, 0.5% BSA, and 0.02% sodium azide . This formulation enhances stability during freeze-thaw cycles.

• Avoid Repeated Freeze-Thaw Cycles: Minimize repeated freezing and thawing as this can lead to denaturation and loss of antibody activity .

• Aliquoting: For frequent users, it is recommended to prepare small aliquots of the antibody to avoid repeated freeze-thaw cycles of the stock solution.

• Working Dilution Preparation: When preparing working dilutions, use freshly prepared buffers and maintain cold conditions (4°C) to preserve antibody activity.

What dilution ranges are recommended for different experimental applications?

Important considerations:

• Initial experiments should begin with the manufacturer's recommended dilution range and then be optimized based on signal intensity and background levels.

• Different cell types and treatments may require adjustment of antibody concentrations for optimal results.

• When detecting proteins with low expression levels or weak phosphorylation signals, lower dilutions (higher antibody concentrations) may be required .

How does antibody specificity for phosphorylated versus non-phosphorylated TP63 compare?

ELISA data demonstrates that Phospho-TP63 (Ser395) antibody exhibits high specificity for the phosphorylated peptide compared to the non-phosphorylated counterpart . The key aspects of phospho-specificity include:

• Validation Method: ELISA tests show that the antibody binds strongly to phospho-peptides containing the phosphorylated Ser395 residue but shows minimal reactivity with the corresponding non-phosphorylated peptide .

• Specificity Statement: According to product documentation, "Phospho-p63 (S395) Polyclonal Antibody detects endogenous levels of p63 protein only when phosphorylated at S395" .

• Immunogen Design: The antibodies are generated against carefully synthesized phospho-peptides specifically designed to represent the region around Ser395 of human p63 (amino acids 361-410) .

• Affinity Purification: The antibodies undergo affinity purification using epitope-specific immunogens to enhance their specificity for the phosphorylated form .

What are the best validation practices for phosphorylation-specific signals in Western blot experiments?

To ensure reliable and reproducible results when using Phospho-TP63 (Ser395) antibody in Western blot experiments, researchers should implement these validation strategies:

• Phosphatase Controls: Treat a portion of your lysate with lambda phosphatase to remove phosphate groups. The phospho-specific signal should disappear in the treated sample while remaining in untreated controls.

• Stimulation/Inhibition Experiments: Treat cells with compounds known to affect the specific phosphorylation pathway. For p63, UV irradiation and serum starvation can modulate phosphorylation status .

• Peptide Competition Assays: Pre-incubate the antibody with excess phospho-peptide (immunogen) before Western blotting. The specific signal should be blocked or significantly reduced.

• Molecular Weight Verification: Confirm that the detected band aligns with the expected molecular weight of p63 (approximately 77 kDa as noted in the product data) .

• Positive and Negative Controls: Include cell lysates known to express or lack phosphorylated p63 at Ser395. Additionally, use an antibody against total p63 to normalize phosphorylation signals.

• Detection Method Optimization: Adjust exposure times and detection methods to optimize signal-to-noise ratios, particularly for low-abundance phosphorylated proteins.

How can Phospho-TP63 (Ser395) Antibody be used to investigate p63 signaling pathways?

Researchers can employ Phospho-TP63 (Ser395) antibody to elucidate p63-mediated signaling networks through several methodological approaches:

• Stimulus-Response Studies: Investigate how various stimuli (UV radiation, DNA damage, growth factors) affect Ser395 phosphorylation of p63 over time. This temporal profiling can reveal pathway kinetics and activation thresholds.

• Kinase Inhibitor Screens: Identify the responsible kinase(s) by systematically treating cells with various kinase inhibitors and monitoring Ser395 phosphorylation levels. This approach can map the upstream regulators of p63.

• Correlation with Functional Outcomes: Combine phosphorylation detection with functional assays (gene expression, cell proliferation, apoptosis) to establish relationships between Ser395 phosphorylation and cellular outcomes.

• Co-Immunoprecipitation Studies: Use the phospho-specific antibody to pull down phosphorylated p63 and identify interacting partners specific to this phosphorylated form, revealing phosphorylation-dependent protein interactions.

• Cell-Based ELISA Screening: Employ cell-based ELISA methods to screen compound libraries for modulators of p63 phosphorylation, potentially identifying novel pathway components .

• Isoform-Specific Effects: Investigate how Ser395 phosphorylation differentially affects various p63 isoforms, given that alternative splicing generates multiple variants with distinct functions .

What considerations should be made when studying TP63 phosphorylation across different cell types?

When investigating p63 phosphorylation in diverse cellular contexts, researchers should consider:

• Baseline Phosphorylation Levels: Different cell types may exhibit varying basal levels of Ser395 phosphorylation, requiring adjusted experimental designs and antibody concentrations.

• Isoform Expression Patterns: Cell types differ in their p63 isoform expression profiles. Since some isoforms may lack certain domains or regulatory regions, interpretation of phosphorylation data must account for the specific isoforms present.

• Cell Culture Conditions: Serum components, cell density, and culture duration can all affect phosphorylation status. Standardize these conditions across experiments and include appropriate controls.

• Tissue-Specific Signaling Networks: The kinases and phosphatases regulating p63 Ser395 phosphorylation may vary between tissues. Context-specific validation is essential.

• Cross-Talk with Other Modifications: Consider how other post-translational modifications of p63 (sumoylation, ubiquitination) might interact with Ser395 phosphorylation . These modifications can be tissue-specific and affect antibody accessibility to the phosphorylation site.

• Normalization Strategies: When comparing phosphorylation levels across cell types, normalize to total p63 levels rather than housekeeping proteins alone to account for varying p63 expression levels.

How can researchers troubleshoot common issues with Phospho-TP63 (Ser395) Antibody experiments?

When encountering challenges in phospho-TP63 detection, consider these methodological solutions:

Challenge 1: Weak or Absent Signal

• Decrease antibody dilution (use more concentrated antibody)

• Increase protein loading amount

• Enhance signal using more sensitive detection systems

• Confirm phosphorylation status by using activators known to induce Ser395 phosphorylation

• Ensure the phosphorylation is preserved by using fresh phosphatase inhibitors in all buffers

Challenge 2: High Background or Non-specific Bands

• Increase antibody dilution

• Extend blocking time or try alternative blocking reagents

• Add 0.1-0.5% Tween-20 to wash buffers and antibody diluents

• Perform additional wash steps

• Pre-adsorb antibody with non-specific proteins

• Optimize transfer conditions for Western blots

Challenge 3: Inconsistent Results Between Experiments

• Standardize cell culture conditions and harvesting procedures

• Create master mixes of antibody dilutions

• Establish positive control lysates that can be included in each experiment

• Document lot-to-lot variations and maintain consistency when possible

• Implement quantitative controls for normalization between experiments

Challenge 4: Discrepancies Between Assay Types

• Different detection methods have varying sensitivities and limitations

• Western blot may show bands at unexpected molecular weights due to post-translational modifications

• ELISA may detect denatured epitopes differently than Western blot

• Validate findings using multiple techniques and approaches

How does p63 phosphorylation relate to its functional roles in cellular processes?

Phosphorylation of p63 at Ser395 represents a critical regulatory mechanism that modulates its activity in various cellular contexts:

• Transcriptional Regulation: Phosphorylation can alter p63's ability to function as a transcriptional activator or repressor. Some p63 isoforms activate RIPK4 transcription, and phosphorylation may modulate this activity .

• Apoptotic Pathways: Phosphorylated p63 may be required in conjunction with p73 for the initiation of p53-dependent apoptosis in response to genotoxic stress and activated oncogenes .

• Notch Signaling Integration: p63 is involved in Notch signaling, potentially through induction of JAG1 and JAG2. Phosphorylation status may influence this cross-talk between signaling pathways .

• Epithelial Morphogenesis: Given p63's role in epithelial development, Ser395 phosphorylation may regulate aspects of morphogenesis and differentiation in epithelial tissues .

• Protein Stability Regulation: Phosphorylation may influence p63's interaction with proteins involved in ubiquitination and sumoylation, thereby affecting its stability and turnover rate .

What emerging technologies are enhancing phosphorylation-specific antibody research?

Several technological advances are improving phospho-specific antibody applications:

• Cell-Based ELISA Techniques: These allow for high-throughput screening of compounds affecting phosphorylation status in intact cells, enabling more physiologically relevant studies .

• Quantitative Phosphoproteomics: Combining immunoprecipitation using phospho-specific antibodies with mass spectrometry enables comprehensive mapping of phosphorylation networks.

• Single-Cell Western Blotting: This technique permits analysis of phosphorylation heterogeneity within cell populations that might be masked in conventional Western blots.

• Tissue-Clearing Methods: These approaches allow for 3D visualization of phosphorylated proteins in intact tissues using phospho-specific antibodies and advanced microscopy.

• Proximity Ligation Assays: These methods enable detection of protein-protein interactions specific to the phosphorylated form of p63, revealing phosphorylation-dependent interactomes.

• CRISPR-Based Phosphorylation Site Editing: Precise genome editing to mutate Ser395 to phosphomimetic (e.g., S395D) or non-phosphorylatable (e.g., S395A) residues allows functional validation of antibody specificity and phosphorylation site function.

How can researchers design experiments to investigate cross-talk between different p63 post-translational modifications?

To study the interplay between phosphorylation at Ser395 and other post-translational modifications of p63, researchers can implement these experimental approaches:

• Sequential Immunoprecipitation: First immunoprecipitate with phospho-Ser395 antibody, then probe for other modifications (ubiquitination, sumoylation) or vice versa to determine co-occurrence.

• Site-Directed Mutagenesis: Generate p63 mutants with altered phosphorylation sites and examine effects on other modifications. For example, does a S395A mutation affect sumoylation or ubiquitination patterns?

• Pharmacological Manipulation: Use inhibitors of specific pathways (kinase inhibitors, deubiquitinase inhibitors) to modulate one modification and observe effects on others.

• Temporal Analysis: Track the sequence of modifications following a stimulus by collecting samples at multiple time points and analyzing with modification-specific antibodies.

• In Vitro Reconstitution: Use purified enzymes to determine if prior phosphorylation enhances or inhibits subsequent modifications in a controlled system.

• Bioinformatic Analysis: Apply computational approaches to predict potential cross-talk based on structural proximity of modification sites and known protein interaction domains.

What are the detailed specifications for available Phospho-TP63 (Ser395) antibodies?

This comprehensive table summarizes the key specifications for both polyclonal and monoclonal variants of Phospho-TP63 (Ser395) antibodies available to researchers .

What controls should be included in Phospho-TP63 (Ser395) antibody experiments?

For rigorous experimental design, researchers should include these controls when working with Phospho-TP63 (Ser395) antibodies:

• Positive Controls:

  • GAPDH detection as an internal loading control for normalization of optical density values

  • Total p63 antibody staining to normalize phospho-specific signals to total protein levels

  • Cell lines known to express phosphorylated p63 (e.g., following appropriate stimulation)

• Negative Controls:

  • Secondary antibody only (omitting primary antibody) to assess non-specific binding

  • HRP-conjugated anti-rabbit IgG antibody alone in different wells (without primary antibodies)

  • Cell lines known to lack p63 expression

  • Phosphatase-treated samples to remove phosphorylation

• Treatment Controls:

  • UV exposure and serum starvation can be used to modulate p63 phosphorylation status

  • Kinase inhibitors that target pathways involved in p63 phosphorylation

• Replicate Samples:

  • Each experimental condition should be performed in duplicate or triplicate for statistical validity

What specialized assay formats are available for studying Phospho-TP63 (Ser395)?

Researchers have access to several specialized assay formats optimized for Phospho-TP63 (Ser395) detection:

• Colorimetric Cell-Based ELISA: This format allows detection of phosphorylated p63 directly in cultured cells without the need for cell lysis or protein extraction. The assay can detect phospho-p63 in as few as 5,000 cells and is suitable for high-throughput screening .

• Normalization Methods for Cell-Based ELISA:

  • Anti-GAPDH antibody for internal positive control normalization

  • Crystal Violet whole-cell staining for cell density normalization

  • Non-phosphorylated p63 antibody for normalizing to total p63 levels

• Phospho-Peptide Competition Assays: These assays confirm antibody specificity by demonstrating signal reduction when the antibody is pre-incubated with the immunogenic phospho-peptide .

• Multiple Detection Systems:

  • HRP-conjugated secondary antibodies for colorimetric detection

  • Fluorescent secondary antibodies for increased sensitivity and multiplexing capabilities

• Cell Treatment Options:

  • Inhibitors, activators, or stimulators to modulate phosphorylation

  • UV exposure and serum starvation as physiological modulators of p63 phosphorylation