DAXX Antibody, HRP conjugated

What is DAXX protein and why is it a significant target for immunodetection?

DAXX (Death domain-associated protein) is a multifunctional nuclear protein with critical roles in transcriptional regulation, chromatin remodeling, and protein homeostasis. As a transcription corepressor, it represses the transcriptional potential of several sumoylated transcription factors and down-regulates both basal and activated transcription . Its significance extends to:

• Histone chaperone function: DAXX facilitates deposition of histone H3.3 variant and is a targeting component of the ATRX:DAXX chromatin remodeling complex

• Protein quality control: Recently identified as a novel type of protein-folding enabler that prevents aggregation and can solubilize pre-existing aggregates

• Apoptosis regulation: Originally identified as a Fas-binding protein, DAXX influences TNFRSF6-dependent apoptosis

• p53 pathway modulation: Acts as an adapter in the MDM2-DAXX-USP7 complex, regulating p53 stability

• Viral restriction: Functions as an antiviral factor that inhibits HIV-1 reverse transcription through SIM-dependent interaction with viral components

The diversity of DAXX functions makes it a significant target for research across multiple disciplines, including cancer biology, neurodegenerative disease research, virology, and epigenetics.

What are the advantages of using HRP-conjugated DAXX antibodies over unconjugated primary antibodies?

HRP-conjugated DAXX antibodies offer significant methodological advantages over unconjugated alternatives:

• Simplified workflow: Direct detection eliminates the need for secondary antibody incubation, reducing protocol time by 1-2 hours and minimizing wash steps

• Enhanced sensitivity: Direct conjugation provides excellent signal-to-noise ratios for detecting low-abundance DAXX protein, particularly important when evaluating nuclear subcompartments

• Reduced cross-reactivity: Eliminates potential cross-species reactivity issues that can occur with secondary antibodies, particularly valuable in multi-species research contexts

• Improved reproducibility: Consistent antibody:enzyme ratio in each experiment reduces inter-assay variability

• Multiplexing capability: Compatible with multi-color detection schemes when combined with other directly labeled antibodies using different detection systems

How should DAXX antibody, HRP conjugated samples be stored to maintain optimal activity?

Proper storage is critical for maintaining the dual functionality of both the antibody binding capacity and HRP enzymatic activity. Based on manufacturer recommendations and research protocols:

Researchers should avoid buffer components that may interfere with HRP activity, including thimerosal, merthiolate, sodium azide at >0.1%, and reducing agents like DTT or mercaptoethanol .

What considerations are important when selecting the appropriate DAXX antibody, HRP conjugated for specific applications?

Selecting the optimal DAXX antibody requires careful consideration of several experimental parameters:

• Epitope location and accessibility: DAXX contains functional domains including SIM (SUMO-interacting motif) and histone-binding regions. Antibodies targeting different epitopes may yield different results based on protein interactions or conformational states

• Species cross-reactivity: While DAXX is highly conserved, confirm reactivity with your target species. Available antibodies show validated reactivity with human samples , but mouse and rat reactivity varies by clone

• Application-specific validation:

  • For Western blotting: Select antibodies validated to detect the 100-130 kDa DAXX band

  • For IHC/ICC: Choose antibodies validated for proper nuclear localization pattern in PML bodies

  • For IP applications: Select antibodies specifically validated for immunoprecipitation of native DAXX

• Clone type considerations:

  • Monoclonal options (e.g., clone E94 ) provide high specificity

  • Polyclonal options may offer enhanced detection across multiple epitopes

• DAXX molecular weight detection: Note that while calculated MW is 81 kDa, observed MW is typically 100-130 kDa due to post-translational modifications

What are the optimal dilution ranges for DAXX antibody, HRP conjugated in different applications?

Appropriate dilution is critical for balancing signal strength and specificity. Based on validated protocols:

Researchers should note that sample-specific factors may necessitate optimization, and preliminary titration experiments are recommended to determine optimal dilution for each experimental system .

What are the most effective blocking conditions for minimizing background when using DAXX antibody, HRP conjugated?

Effective blocking is essential for minimizing non-specific binding and optimizing signal-to-noise ratio:

• Protein blockers:

  • 5-10% non-fat dry milk in TBST is effective for Western blotting applications with DAXX antibodies

  • 10% goat serum shows excellent results for IHC/ICC applications with DAXX antibodies

  • BSA (1-5%) may be preferred when phospho-specific DAXX detection is needed

• Block optimization by application:

  • For Western blotting: 5% non-fat milk in TBST for 1 hour at room temperature

  • For IHC: 10% goat serum in PBS for 30-60 minutes at 37°C, as validated in rat intestine and human intestinal cancer tissue samples

  • For ICC: Blocking with 10% goat serum shows excellent results with SMMC-7721 and A549 cells

• Background reduction strategies:

  • Extend washing steps (5-6 washes of 5 minutes each) when high background is observed

  • Inclusion of 0.1-0.3% Triton X-100 in blocking buffer can reduce non-specific hydrophobic interactions

  • Pre-adsorption of antibody with non-relevant tissues can reduce non-specific binding

How can researchers address variability in DAXX detection between nuclear and cytoplasmic fractions?

DAXX predominantly localizes to the nucleus but can translocate to the cytoplasm under stress conditions, creating detection challenges:

• Subcellular fractionation approach:

  • Use specialized nuclear extraction buffers containing 420 mM NaCl to efficiently extract DAXX from nuclear bodies

  • Sequential extraction protocols starting with cytoplasmic lysis (0.1% NP40) followed by nuclear extraction yield cleaner fraction separation

  • Confirm fraction purity using nuclear (TBP ) and cytoplasmic (GAPDH) markers in parallel

• Fixation considerations for microscopy:

  • Paraformaldehyde (4%) fixation for 15 minutes preserves DAXX nuclear structure

  • For detection of cytoplasmic DAXX under stress conditions, shorter fixation times (5-10 minutes) may better preserve cytoplasmic signal

  • Methanol fixation can sometimes improve nuclear DAXX detection in PML bodies

• Stress-induced translocation monitoring:

  • When studying stress-induced DAXX translocation, include time-course analysis

  • DAXX shows translocation to the cytoplasm under conditions of stress and activates the JNK pathway

• Antibody selection for subcellular pools:

  • Some antibody clones may preferentially detect nuclear or cytoplasmic DAXX pools

  • Validation with both immunofluorescence and subcellular fractionation Western blotting is recommended

What strategies can overcome detection challenges when DAXX is present at low abundance or complexed with other proteins?

DAXX detection can be challenging due to its involvement in multiple protein complexes and varying expression levels:

• Enhanced chemiluminescence approaches:

  • Use high-sensitivity ECL substrates with extended exposure times (up to 5 minutes)

  • Super-signal West Femto or similar ultra-sensitive detection reagents can improve detection of low-abundance DAXX

• Protein complex disruption strategies:

  • Sample denaturation at 95°C for 10 minutes in SDS sample buffer containing 8M urea can disrupt strong protein complexes

  • Consider sonication (3 × 10s pulses) of samples to improve release of DAXX from nuclear bodies

• Signal enhancement techniques:

  • Tyramide signal amplification can significantly enhance HRP-conjugated antibody signals in IHC/ICC applications

  • For Western blotting, nitrocellulose membranes often provide better sensitivity than PVDF for DAXX detection

• Epitope retrieval optimization:

  • For formalin-fixed tissues, extended antigen retrieval (25-30 minutes) with TE buffer (pH 9.0) significantly improves DAXX detection

  • Enzyme-based antigen retrieval shows excellent results for detection in cell lines, as demonstrated with SMMC-7721 and A549 cells

How does post-translational modification affect DAXX detection, and how can researchers account for these variations?

DAXX undergoes extensive post-translational modifications that affect its detection pattern:

• Molecular weight variation:

  • DAXX has a calculated molecular weight of 81 kDa but typically migrates at 100-130 kDa due to post-translational modifications

  • SUMOylation can add approximately 11-14 kDa per SUMO group

  • Phosphorylation further alters migration patterns

• Detection of modified DAXX:

  • SUMOylated DAXX is critical for function, and the SUMO-interacting motif (SIM) is required for DAXX-mediated inhibition of HIV-1 reverse transcription

  • When studying SUMOylated DAXX, include NEM (N-ethylmaleimide, 20mM) in lysis buffers to preserve SUMO modifications

  • Phosphorylated DAXX detection may require phosphatase inhibitor cocktails in sample preparation buffers

• Proteasomal degradation considerations:

  • DAXX undergoes proteasome-dependent degradation by viral E1B-55K

  • Include proteasome inhibitors (e.g., MG132, 10μM for 4-6 hours) in experiments studying DAXX degradation pathways

• Antibody selection for modified forms:

  • Different antibody clones may have differential reactivity with modified DAXX forms

  • Epitope masking due to protein-protein interactions or modifications may require testing multiple antibodies targeting different regions

How can researchers effectively employ DAXX antibody, HRP conjugated in studies of DAXX's molecular chaperone functions?

Recent research has revealed DAXX as a novel molecular chaperone. To investigate this function:

• Experimental design for protein aggregation studies:

  • DAXX prevents aggregation of model substrates like luciferase and neurodegeneration-associated proteins

  • DAXX can solubilize pre-existing aggregates and unfold misfolded proteins

  • Protocols should include heat-stress conditions (42°C) for model substrates to induce aggregation

• Key methodological approaches:

  • Light scattering assays to monitor DAXX's ability to prevent protein aggregation

  • Luciferase enzymatic activity assays to assess DAXX's protection of functional integrity

  • Filter trap assays to quantify DAXX's disaggregation of amyloid proteins like Aβ42

• Disease-associated protein studies:

  • DAXX has shown differential activity toward different aggregates: effective with ATXN1(82Q) and Aβ42 fibrils but not α-Syn fibrils

  • Bimolecular fluorescence complementation (BiFC) systems can be used to detect oligomeric intermediates in cells

• Cellular validation approaches:

  • Co-expression of DAXX with structurally destabilized proteins (e.g., nLucDM) demonstrates its chaperone function

  • DAXX reduces the size and number of ATXN1(82Q) inclusions more effectively than HSP70 in cellular models

What approaches can investigate DAXX's role in the regulation of p53 and MDM2 pathways using HRP-conjugated antibodies?

DAXX plays a critical role in p53-MDM2 regulatory networks. To investigate this function:

• Biochemical approaches:

  • DAXX maintains the native conformation of both p53 and MDM2

  • Thermal shift assays demonstrate DAXX's ability to stabilize p53's native conformation

  • In vitro ubiquitination assays show DAXX enhances MDM2-mediated p53 ubiquitination

• Cellular pathway analysis:

  • DAXX reduces p53 protein levels in U2OS cells

  • In H1299 cells with inducible p53 expression, DAXX decreases p53 levels and lowers expression of p53 target genes

• Co-immunoprecipitation studies:

  • Use DAXX antibody for pull-down followed by immunoblotting for MDM2, USP7, and p53

  • Under non-stress conditions, DAXX associates with MDM2 and USP7

  • Upon DNA damage, this association is disrupted, leading to p53 stabilization

• Experimental considerations:

  • Include appropriate controls for stress conditions (DNA damage inducers like etoposide)

  • Monitor p53 target gene expression by RT-qPCR to assess functional outcomes

  • Consider the dynamic nature of these interactions across different cellular states

How can DAXX antibody, HRP conjugated be applied in studying DAXX's role in histone variant deposition and chromatin remodeling?

DAXX functions as a histone chaperone for H3.3 variant deposition, which can be studied using these approaches:

• Chromatin immunoprecipitation (ChIP) strategies:

  • DAXX antibodies can be used to identify genomic regions where DAXX facilitates H3.3 deposition

  • Special attention to pericentric repeats, telomeres, and regulatory elements of immediate early genes is recommended

• Protein complex analysis:

  • DAXX and ATRX form a chromatin remodeling complex with ATP-dependent DNA translocase activity

  • Co-immunoprecipitation with DAXX antibody followed by detection of ATRX and histone H3.3 can identify these complexes

  • Recent research indicates the DAXX-ATRX interaction is dispensable for viability while the DAXX-H3.3 interaction is essential for postnatal viability

• Mutation impact studies:

  • Two key mutations have been developed to study DAXX function: Daxx^Y130A^ (abolishes DAXX-ATRX interaction) and Daxx^S226A^ (abolishes DAXX-H3.3 interaction)

  • Transcriptome analysis reveals these interactions are important for silencing endogenous retroviruses

• Experimental design considerations:

  • Include chromatin fractionation steps to effectively extract DAXX from chromatin

  • Consider crosslinking approaches for stabilizing transient interactions

  • Analyze both global H3.3 deposition patterns and specific genomic loci related to DAXX function

What methodological approaches can investigate DAXX's antiviral functions, particularly in HIV-1 restriction?

Recent research has identified DAXX as an antiretroviral factor that inhibits HIV-1 replication:

• Key mechanistic insights:

  • DAXX inhibits HIV-1 reverse transcription through a SIM-dependent interaction with cyclophilin A (CypA) and viral capsid (CA)

  • DAXX expression is upregulated by interferon in both murine and human cells

  • The SIM domain is required for DAXX-mediated inhibition of reverse transcription

• Experimental approaches:

  • RT-qPCR analyses to quantify viral reverse transcription products in DAXX-expressing versus DAXX-depleted cells

  • Proteomic screening (SILAC coupled with LC-MS/MS) to identify DAXX-interacting proteins during viral infection

  • Biochemical analyses to study DAXX binding to incoming HIV-1 cores

• Mutation-based studies:

  • Use DAXX SIM mutants to demonstrate the requirement of SUMO-interaction for antiviral activity

  • Determine if DAXX's target (viral or cellular) is SUMOylated

• DAXX-mediated restriction assessment:

  • Measure enhanced virus growth, synthesis of early viral proteins, viral mRNA, and DNA in DAXX-depleted human cells

  • Analyze E1B-55K-mediated proteasomal degradation of DAXX via cullin-5-dependent E3-ubiquitin-ligase in adenovirus infection

What emerging techniques might enhance the utility of DAXX antibody, HRP conjugated in primary research?

Several innovative approaches show promise for expanding DAXX research capabilities:

• Multiplexed imaging technologies:

  • Cyclic immunofluorescence (CycIF) allows sequential imaging of multiple targets including DAXX

  • Mass cytometry imaging (IMC) could provide simultaneous detection of DAXX alongside dozens of other proteins

• Proximity labeling approaches:

  • BioID or APEX2 fusions with DAXX can identify proximal proteins in living cells

  • HRP-conjugated DAXX antibodies could be adapted for proximity labeling of DAXX-associated proteins in fixed samples

• Single-molecule detection systems:

  • Super-resolution microscopy combined with HRP-based signal amplification could reveal nanoscale DAXX distribution

  • Live-cell single-molecule tracking could monitor DAXX dynamics during stress responses

• Microfluidic applications:

  • Automated microfluidic immunostaining platforms could enhance reproducibility of DAXX detection

  • Single-cell proteomic approaches might reveal heterogeneity in DAXX expression and modification states

How might DAXX antibody, HRP conjugated contribute to understanding neurodegenerative disease mechanisms?

DAXX's recently discovered chaperone functions suggest important applications in neurodegeneration research:

• Protein aggregation disease models:

  • DAXX has shown activity against amorphous ATXN1(82Q) aggregates and Aβ42 fibrils

  • Investigation of DAXX levels and activity in Alzheimer's, Huntington's, and ataxia models could reveal disease mechanisms

• Therapeutic potential assessment:

  • Monitoring DAXX expression and localization in response to candidate drugs

  • Screening compounds that might enhance DAXX's chaperone activity

• Genotype-phenotype correlations:

  • Correlating DAXX levels with disease progression in patient samples

  • Investigating DAXX polymorphisms as potential disease modifiers

• Mechanistic dissection:

  • Detailed analysis of how DAXX disaggregates Aβ42 fibrils but not α-Syn fibrils

  • Investigation of potential synergies between DAXX and other chaperone systems