Phospho-DAXX (Ser739) Antibody

What is Phospho-DAXX (Ser739) Antibody and what epitope does it recognize?

Phospho-DAXX (Ser739) Antibody is a rabbit polyclonal antibody specifically designed to recognize the DAXX protein when it is phosphorylated at serine residue 739. The antibody targets the peptide sequence L-S-D-S(p)-D derived from Human DAXX surrounding the phosphorylated serine 739 . This high specificity allows researchers to monitor the phosphorylation status of DAXX at this particular site, which can be indicative of certain cellular signaling pathways or protein activation states. Unlike general DAXX antibodies, this phosphospecific antibody enables the precise detection of this post-translational modification, providing critical information about the functional state of the protein in various experimental conditions .

What experimental applications has the Phospho-DAXX (Ser739) Antibody been validated for?

The Phospho-DAXX (Ser739) Antibody has been validated for multiple experimental applications, with primary validation in enzyme-linked immunosorbent assay (ELISA) and Western blot (WB) techniques . For Western blot applications, the recommended dilution range is 1:500-1:1000, which allows for optimal signal-to-noise ratio while preserving antibody resources . This antibody enables researchers to detect changes in DAXX phosphorylation status in response to various stimuli, cellular stresses, or pharmacological interventions. While not explicitly validated for immunohistochemistry or flow cytometry in the provided data, phosphospecific antibodies as a class are frequently employed in these applications as well, suggesting potential broader utility with proper optimization .

What species reactivity does the Phospho-DAXX (Ser739) Antibody exhibit?

The Phospho-DAXX (Ser739) Antibody demonstrates confirmed reactivity across three mammalian species: human, mouse, and rat . This cross-species reactivity is particularly valuable for comparative studies examining DAXX phosphorylation mechanisms across different model systems. The conservation of reactivity suggests that the region surrounding Ser739 may be evolutionarily conserved across these species, indicating potential functional significance of this phosphorylation site. Researchers should note that while these three species have been validated, the antibody may potentially recognize DAXX phosphorylation in other closely related mammalian species, though additional validation would be required before use in such contexts .

What are the optimal storage and handling conditions for Phospho-DAXX (Ser739) Antibody?

The Phospho-DAXX (Ser739) Antibody requires specific storage conditions to maintain its activity and specificity. Upon receipt, the antibody should be stored at either -20°C or -80°C to preserve functionality . The antibody is supplied in a stabilizing buffer formulation consisting of phosphate buffered saline (without Mg²⁺ and Ca²⁺), pH 7.4, containing 150mM NaCl, 0.02% sodium azide, and 50% glycerol at a concentration of 1.0mg/mL . This formulation helps maintain antibody stability during freeze-thaw cycles, though repeated freeze-thaw cycles should be avoided to prevent degradation. When working with the antibody, researchers should aliquot the stock solution into smaller volumes to minimize freeze-thaw cycles, maintain cold chain procedures during handling, and return the antibody to cold storage promptly after use to ensure optimal performance in experimental applications .

How should researchers validate the specificity of the Phospho-DAXX (Ser739) Antibody in their experimental system?

Validating the specificity of Phospho-DAXX (Ser739) Antibody requires a multi-step approach:

• Phosphopeptide Competition Assay: Researchers should perform Western blot analyses with and without pre-incubation of the antibody with the specific phosphopeptide used as the immunogen. Specific signal should be blocked by the phosphopeptide but not by the corresponding non-phosphorylated peptide .

• Phosphatase Treatment Control: Treating one sample set with lambda phosphatase before antibody probing should eliminate the signal if the antibody is truly phospho-specific .

• Stimulation/Inhibition Experiments: Treating cells with agents known to increase or decrease phosphorylation at Ser739 can demonstrate dynamic changes in signal intensity correlating with treatment conditions.

• Site-Directed Mutagenesis: Similar to validation methods used for other phospho-antibodies, expressing a DAXX S739A mutant should result in loss of signal compared to wild-type DAXX .

• siRNA Knockdown: Confirming signal loss when DAXX is knocked down using siRNA provides additional evidence of specificity, as demonstrated with other phospho-DAXX antibodies .

These methods collectively provide robust validation of phospho-specificity, ensuring reliable experimental results when using this antibody.

What controls should be included when using Phospho-DAXX (Ser739) Antibody in Western blot experiments?

When conducting Western blot experiments with Phospho-DAXX (Ser739) Antibody, researchers should implement the following controls to ensure experimental validity:

• Positive Control: Include a lysate from cells known to exhibit DAXX Ser739 phosphorylation, such as certain stimulated cell lines.

• Negative Control: One approach is to use lambda phosphatase-treated samples to remove phosphate groups, which should eliminate or significantly reduce the signal.

• Loading Control: Include antibodies against housekeeping proteins (e.g., GAPDH, β-actin) to normalize for total protein loading variations.

• Total DAXX Control: Run parallel blots or strip and reprobe with an antibody recognizing total DAXX regardless of phosphorylation status to determine whether observed changes reflect altered phosphorylation or altered total protein levels.

• Peptide Competition Control: Pre-incubate antibody with the phosphopeptide immunogen to demonstrate signal specificity, similar to verification methods used for other phospho-specific antibodies .

• Molecular Weight Marker: Include a molecular weight ladder to confirm that the detected band corresponds to the expected size of DAXX (approximately 120 kDa).

These controls collectively help distinguish between true phosphorylation events and potential artifacts or non-specific binding.

What is the functional significance of DAXX phosphorylation at Ser739?

While the specific functional significance of DAXX phosphorylation at Ser739 is not explicitly detailed in the provided search results, we can draw inferences based on the general functions of DAXX and known mechanisms of phosphorylation at other sites. DAXX functions as a transcription corepressor that represses the transcriptional potential of several sumoylated transcription factors and down-regulates both basal and activated transcription . Its activity is modulated through recruitment to subnuclear compartments like nucleoli or PML/POD/ND10 nuclear bodies via interactions with proteins like MCSR1 and PML .

Phosphorylation often serves as a regulatory mechanism for protein-protein interactions and subcellular localization. By analogy with the well-characterized Ser564 phosphorylation (which disrupts the DAXX-MDM2 interaction upon DNA damage ), Ser739 phosphorylation may similarly regulate DAXX interactions with its binding partners, potentially affecting:

• Transcriptional repression activities

• Interaction with sumoylated transcription factors

• Localization to nuclear bodies

• Involvement in apoptosis pathways, particularly TNFRSF6-dependent processes

Research focusing specifically on the Ser739 phosphorylation site would be needed to determine its precise regulatory role in DAXX function.

How does DAXX phosphorylation fit into broader cellular signaling networks?

DAXX phosphorylation represents an important node within broader cellular signaling networks, particularly those involved in transcriptional regulation, stress response, and apoptosis. Based on the available information on DAXX function and phosphorylation mechanisms:

• Transcriptional Regulation Network: DAXX serves as a transcription corepressor that inhibits the transcriptional activation of multiple factors including PAX3 and ETS1 through direct protein-protein interactions . Phosphorylation likely modulates these interactions, integrating signals from various cellular pathways into transcriptional outputs.

• MDM2-DAXX-USP7 Complex: DAXX functions as an adapter protein in this complex, regulating the RING-finger E3 ligase MDM2 . By analogy with Ser564 phosphorylation, which disrupts the DAXX-MDM2 interaction during DNA damage response , Ser739 phosphorylation may similarly affect protein complex formation and stability.

• PML Nuclear Body Dynamics: DAXX localizes to PML/POD/ND10 nuclear bodies and regulates transcription in conjunction with PML . Phosphorylation events may control this localization pattern, connecting kinase signaling pathways with subnuclear compartmentalization.

• Apoptosis Signaling: DAXX may influence TNFRSF6-dependent apoptosis , with phosphorylation potentially serving as a molecular switch that determines pro-survival versus pro-apoptotic functions.

The specific kinases targeting Ser739 remain to be fully characterized, unlike ATM which phosphorylates Ser564 during DNA damage response .

How does the phosphorylation pattern of DAXX differ across cell types and under various stimuli?

The phosphorylation pattern of DAXX demonstrates considerable plasticity across different cell types and in response to various stimuli, reflecting its role in integrating diverse cellular signals. While the search results don't provide comprehensive data specifically for Ser739 phosphorylation across cell types, we can make several observations:

• Cell Type Variation: Phosphorylation of DAXX has been observed across multiple cell lines with varying p53 status, including both p53-wild-type cells (U2OS, HT1080, IMR90) and p53-deficient cells (H1299, Saos-2, HeLa, 293T), suggesting that some DAXX phosphorylation events occur independently of p53 status . This indicates cell-type-specific regulation may exist for DAXX phosphorylation.

• Response to DNA Damage: For the well-characterized Ser564 site, phosphorylation occurs rapidly (within 10 minutes) following treatment with DNA-damaging agents like etoposide or ionizing radiation, and this phosphorylation precedes p53 activation in p53-wild-type cells . By analogy, Ser739 might also show dynamic phosphorylation patterns in response to specific cellular stresses.

• Temporal Dynamics: DAXX phosphorylation can exhibit distinct temporal patterns, with some modifications appearing rapidly after stimulus and persisting for extended periods (over 8 hours for Ser564 phosphorylation after etoposide treatment) .

• Stimulus Specificity: Different stimuli likely induce distinct phosphorylation patterns. For instance, while DNA damage induces Ser564 phosphorylation, other stresses such as hypoxia, osmotic stress, or growth factor signaling might trigger phosphorylation at other sites, potentially including Ser739.

Researchers interested in Ser739 phosphorylation specifically should consider performing phosphorylation profiling across cell types and stimuli using the Phospho-DAXX (Ser739) Antibody.

How can Phospho-DAXX (Ser739) Antibody be used to elucidate the role of DAXX in DNA damage response pathways?

The Phospho-DAXX (Ser739) Antibody can be employed in multiple sophisticated experimental approaches to investigate DAXX's role in DNA damage response pathways:

• Temporal Phosphorylation Profiling: Researchers can track Ser739 phosphorylation dynamics after DNA damage induction using agents like etoposide, ionizing radiation, or UV. This temporal profiling could be compared with the well-characterized Ser564 phosphorylation (which occurs within 10 minutes after DNA damage and precedes p53 activation ) to establish the sequence of phosphorylation events on DAXX.

• Kinase Inhibitor Studies: By treating cells with specific kinase inhibitors prior to DNA damage induction, researchers can identify the kinase(s) responsible for Ser739 phosphorylation. This approach helped establish ATM as the kinase for Ser564 .

• Phosphorylation-Deficient Mutant Analysis: Creating DAXX S739A mutants and comparing their behavior to wild-type DAXX during DNA damage response can reveal the functional significance of this phosphorylation site, similar to studies with the S564A mutant .

• Protein Interaction Analysis: Co-immunoprecipitation experiments comparing wild-type DAXX versus S739A mutants can identify protein interactions that are specifically regulated by Ser739 phosphorylation during DNA damage.

• Chromatin Association Dynamics: Chromatin immunoprecipitation (ChIP) using the Phospho-DAXX (Ser739) Antibody can reveal whether this phosphorylation affects DAXX association with specific genomic regions following DNA damage.

• Subcellular Localization Studies: Immunofluorescence using this antibody can determine whether Ser739 phosphorylation influences DAXX localization to PML nuclear bodies or other subnuclear structures during DNA damage response.

These approaches collectively can establish whether Ser739 phosphorylation, like Ser564 phosphorylation, contributes to critical functions such as disrupting DAXX-MDM2 interactions and facilitating p53 activation during DNA damage response.

What techniques can be employed to study the interplay between different DAXX phosphorylation sites?

Investigating the interplay between different DAXX phosphorylation sites, such as between Ser739 and the well-characterized Ser564, requires sophisticated methodological approaches:

• Mass Spectrometry-Based Phosphorylation Profiling: Quantitative phosphoproteomics can identify all phosphorylation sites on DAXX simultaneously, enabling analysis of co-occurrence patterns or potential sequential phosphorylation events under various experimental conditions.

• Multi-phosphosite-specific Antibodies: Using combinations of phospho-specific antibodies (including Phospho-DAXX (Ser739) and others like Phospho-DAXX (Ser564)) in Western blot or immunoprecipitation experiments can reveal correlation or mutual exclusivity between different phosphorylation events.

• Phosphomimetic and Phospho-deficient Mutants: Creating DAXX variants with various combinations of mutations (e.g., S564A/S739A double mutant, S564D/S739A, S564A/S739D, S564D/S739D) can help determine whether phosphorylation at one site influences phosphorylation at another site.

• Kinase Inhibitor Studies: Selective inhibition of specific kinases (e.g., ATM which phosphorylates Ser564 ) can reveal whether blocking one phosphorylation event affects others, potentially identifying hierarchical relationships between phosphorylation sites.

• Time-course Analysis: Comparing the temporal dynamics of phosphorylation at different sites following stimulation can identify sequential phosphorylation patterns and potential priming events.

• Proximity Ligation Assays: This technique can detect the co-occurrence of multiple phosphorylation events on the same DAXX molecule, rather than merely within the same cell population.

• Protein Interaction Studies with Phosphosite Mutants: Comparing how different phosphosite mutations affect DAXX interactions with partners like MDM2, USP7, or PML can reveal functional relationships between phosphorylation sites.

These complementary approaches can establish whether Ser739 and Ser564 phosphorylations function cooperatively, antagonistically, or independently in regulating DAXX function.

How can computational approaches complement experimental studies using Phospho-DAXX (Ser739) Antibody?

Computational approaches provide powerful complementary tools to experimental studies employing the Phospho-DAXX (Ser739) Antibody:

• Phosphorylation Site Conservation Analysis: Bioinformatic comparison of DAXX sequences across species can reveal evolutionary conservation of Ser739 and surrounding residues, indicating functional importance. Unlike Ser564, which is conserved in mammalian species but not in fish or flies , understanding Ser739 conservation patterns may provide evolutionary context for this phosphorylation site.

• Kinase Prediction Algorithms: Computational tools can predict potential kinases targeting Ser739 based on consensus sequence analysis, generating testable hypotheses about the signaling pathways regulating this phosphorylation.

• Structural Modeling: Molecular dynamics simulations can predict how Ser739 phosphorylation might alter DAXX protein conformation, potentially affecting interaction surfaces for binding partners.

• Phosphorylation-Dependent Protein Interaction Prediction: Algorithms can predict how phosphorylation at Ser739 might create or disrupt binding motifs for phospho-binding domains (e.g., 14-3-3 proteins, WW domains, FHA domains).

• Network Analysis: Integration of phosphoproteomics data with protein-protein interaction databases can place DAXX Ser739 phosphorylation within broader signaling networks, identifying potential functional connections.

• Machine Learning Approaches: These can identify patterns in large datasets correlating DAXX Ser739 phosphorylation with other cellular events, gene expression patterns, or phenotypic outcomes.

• Molecular Docking: Computational docking studies can predict how Ser739 phosphorylation might affect DAXX interactions with known binding partners like MDM2 or PML.

These computational approaches generate hypotheses that can be experimentally validated using the Phospho-DAXX (Ser739) Antibody, creating a powerful iterative cycle between computational prediction and experimental verification.

What are common technical challenges when using Phospho-DAXX (Ser739) Antibody and how can they be addressed?

Researchers may encounter several technical challenges when working with Phospho-DAXX (Ser739) Antibody. Here are common issues and their solutions:

• High Background Signal:

  • Cause: Insufficient blocking, excessive antibody concentration, or cross-reactivity

  • Solution: Optimize blocking conditions (try different blocking agents like 5% BSA instead of milk for phospho-epitopes), titrate antibody concentration (starting with the recommended 1:500-1:1000 dilution for Western blot ), and increase washing duration and frequency

• Weak or Absent Signal:

  • Cause: Low phosphorylation levels, phosphatase activity during sample preparation, or epitope masking

  • Solution: Include phosphatase inhibitors in lysis buffers, optimize stimulation conditions to increase phosphorylation, reduce sample heating time, and confirm total DAXX expression using a non-phospho-specific antibody

• Multiple Bands or Unexpected Band Size:

  • Cause: DAXX degradation, cross-reactivity, or post-translational modifications

  • Solution: Use fresh samples with protease inhibitors, validate with DAXX knockdown or overexpression controls, and compare with expected molecular weight (approximately 120 kDa)

• Inconsistent Results Between Experiments:

  • Cause: Variable phosphorylation status, antibody degradation, or protocol inconsistencies

  • Solution: Standardize cell treatment protocols, aliquot antibody to avoid freeze-thaw cycles, and include positive controls in each experiment

• Poor Reproducibility Across Cell Types:

  • Cause: Cell-type specific phosphorylation patterns or interfering proteins

  • Solution: Optimize lysis conditions for each cell type and validate antibody specificity in each experimental system

• Signal Loss During Storage:

  • Cause: Antibody degradation or epitope dephosphorylation

  • Solution: Store antibody at recommended temperatures (-20°C or -80°C ), aliquot to minimize freeze-thaw cycles, and add phosphatase inhibitors to lysates

Addressing these challenges through systematic optimization will improve experimental outcomes when using this specialized antibody.

How should researchers interpret conflicting results between phosphorylation detected by antibody-based methods versus mass spectrometry?

When faced with discrepancies between phosphorylation data obtained using the Phospho-DAXX (Ser739) Antibody and mass spectrometry (MS) results, researchers should consider the following interpretive framework:

• Sensitivity Differences: Antibody-based methods often have higher sensitivity for detecting specific phosphorylation events compared to MS, which may miss low-abundance phosphopeptides. If antibody detects phosphorylation not found by MS, consider:

  • Enriching for phosphopeptides before MS analysis

  • Increasing sample amount for MS

  • Using targeted MS approaches for the specific phosphopeptide

• Specificity Considerations: Antibodies may occasionally recognize similar phosphoepitopes or cross-react with other proteins. If MS fails to confirm antibody-detected phosphorylation:

  • Validate antibody specificity using phosphopeptide competition assays

  • Perform additional controls with phosphatase treatment

  • Use site-directed mutants (S739A) as negative controls

  • Consider the possibility that the antibody recognizes a similar epitope on another protein

• Dynamic Range Differences: MS may struggle with detecting phosphorylation on low-abundance proteins, while antibodies can amplify these signals. Consider:

  • Immunoprecipitating DAXX before MS analysis

  • Using absolute quantification standards in MS

• Sample Preparation Effects: Differences in sample preparation between techniques may affect phosphorylation preservation:

  • Standardize phosphatase inhibitor usage across both methods

  • Consider rapid sample processing techniques like heat stabilization

  • Use identical cell stimulation conditions for both approaches

• Confirmatory Approaches: When results conflict, employ additional techniques:

  • Use multiple phospho-specific antibodies recognizing different epitopes

  • Employ Phos-tag™ SDS-PAGE to separate phosphorylated from non-phosphorylated proteins

  • Use in vitro kinase assays with purified components

By systematically addressing these factors, researchers can resolve discrepancies and develop a more complete understanding of DAXX Ser739 phosphorylation dynamics.

What considerations should researchers take into account when analyzing DAXX phosphorylation in different experimental models?

When analyzing DAXX phosphorylation across different experimental models using the Phospho-DAXX (Ser739) Antibody, researchers should consider several important factors:

• Species-Specific Variations:

  • While the antibody shows reactivity with human, mouse, and rat DAXX , sequence variations around Ser739 may affect antibody affinity across species

  • Validate antibody performance in each species by including appropriate positive controls

  • Consider sequence alignment analysis to determine conservation of the Ser739 site across species

• Cell Type-Specific Regulation:

  • Basal phosphorylation levels may vary significantly between cell types

  • Stimuli that induce phosphorylation in one cell type may not be effective in others

  • Expression levels of relevant kinases and phosphatases differ between cell models

  • Consider analyzing both phospho-DAXX and total DAXX to normalize for expression differences

• Culture Condition Effects:

  • Serum components may activate signaling pathways affecting DAXX phosphorylation

  • Cell density and growth phase can influence phosphorylation patterns

  • Standardize culture conditions before comparative analyses

  • Consider serum starvation before stimulation to reduce background phosphorylation

• In Vitro versus In Vivo Models:

  • Phosphorylation patterns observed in cell culture may differ from those in tissue samples

  • For tissue analysis, rapid sample preservation is critical to maintain phosphorylation status

  • Consider tissue-specific extraction protocols optimized for phosphoprotein preservation

• Genetic Background Considerations:

  • Genetic alterations affecting upstream signaling pathways may influence DAXX phosphorylation

  • In transgenic models, confirm that introduced mutations don't affect the phospho-epitope

  • For patient-derived samples, consider how disease-related mutations might affect phosphorylation

• Quantification Approaches:

  • Normalize phospho-DAXX signal to total DAXX rather than housekeeping proteins

  • Consider using phosphorylation standards for absolute quantification

  • For comparative studies, process all samples in parallel to minimize technical variation

By accounting for these variables, researchers can generate more reliable and interpretable data on DAXX Ser739 phosphorylation across different experimental systems.