DAXX Antibody

What is DAXX protein and why is it important in research?

DAXX is a ubiquitously expressed protein originally identified as an interactor with the cytoplasmic domain of Fas . It plays crucial roles in multiple cellular processes:

• Acts as a histone chaperone facilitating deposition of histone H3.3

• Functions in chromatin remodeling as part of the ATRX:DAXX complex

• Mediates activation of the JNK pathway and apoptosis

• Regulates transcription of immediate early genes

• Shows restriction activity towards human cytomegalovirus (HCMV)

Its involvement in these diverse cellular pathways makes DAXX a significant target for researchers studying chromatin regulation, apoptosis, and cancer biology.

While the calculated molecular weight of DAXX is approximately 81 kDa based on its amino acid sequence , it typically appears at higher molecular weights on Western blots:

• Commonly observed at 100-120 kDa

• Sometimes detected at 70 kDa

• Occasionally appears as multiple bands (e.g., 100 kDa and 48 kDa)

This size discrepancy is primarily due to post-translational modifications, particularly phosphorylation. DAXX contains numerous phosphorylation sites that influence its migration pattern on SDS-PAGE. Additionally, alternative splicing may generate different isoforms. When validating a new DAXX antibody, it's essential to use appropriate positive and negative controls (such as DAXX knockout cell lines) to confirm specificity .

How should I optimize DAXX antibody dilutions for different applications?

Optimization of DAXX antibody dilutions is critical for specific signal detection:

For Western Blotting:

• Start with manufacturer's recommended dilution (typically 1:1000)

• Prepare a dilution series (e.g., 1:500, 1:1000, 1:2000, 1:5000)

• Include appropriate positive controls (e.g., HeLa cells, which express DAXX)

• Include negative controls (ideally DAXX knockout cell lines)

• Assess signal-to-noise ratio to determine optimal dilution

For Immunohistochemistry:

• Begin with dilutions between 1:250-1:1000

• Perform antigen retrieval with citrate buffer pH 6.0 or TE buffer pH 9.0

• Titrate antibody concentration to achieve specific nuclear staining

• Include positive tissue controls (e.g., human stomach tissue)

For Immunofluorescence:

• Start with higher concentrations (1:25-1:50)

• Optimize fixation method (4% formaldehyde or 100% methanol)

• Include counterstains to visualize nuclei (DAPI) and cell boundaries

Remember that optimal dilutions may vary between antibody lots and should be determined empirically for each new application or sample type .

What are the best methods for validating DAXX antibody specificity?

Rigorous validation of DAXX antibody specificity is essential for reliable research:

• Genetic approaches:

  • Use DAXX knockout cell lines as negative controls

  • Compare staining patterns in wild-type vs. knockout cells by Western blot, immunofluorescence, or IHC

• Peptide competition assays:

  • Pre-incubate antibody with the immunizing peptide

  • Loss of signal indicates specific binding

• Multiple antibody validation:

  • Use different antibodies targeting distinct DAXX epitopes

  • Consistent results with different antibodies support specificity

• siRNA knockdown:

  • Transfect cells with DAXX-specific siRNA for 72 hours

  • Verify reduced protein expression by Western blot

  • Compare immunofluorescence staining between control and knockdown cells

• Expected localization patterns:

  • DAXX is predominantly nuclear with enrichment in PML nuclear bodies

  • Verify subcellular localization matches known distribution patterns

The gold standard validation combines multiple approaches, particularly genetic knockout/knockdown strategies with biochemical verification .

What antigen retrieval methods are recommended for DAXX immunohistochemistry?

Effective antigen retrieval is crucial for DAXX detection in formalin-fixed, paraffin-embedded tissues:

• Heat-induced epitope retrieval (HIER):

  • Citrate buffer (pH 6.0): Heat for 20 minutes at 98°C

  • TE buffer (pH 9.0): Alternative method for some antibodies

• Protocol optimization:

  • Tissue fixation affects antigen accessibility; optimize retrieval time based on fixation duration

  • Compare different retrieval buffers for your specific tissue type

  • Ensure complete deparaffinization before antigen retrieval

• Considerations for specific tissues:

  • For stomach tissue, citrate buffer (pH 6.0) has been validated

  • For bladder urothelial carcinoma, HIER with citrate buffer for 20 min at 98°C shows good results

• Post-retrieval processing:

  • Block endogenous peroxidase activity with 3% hydrogen peroxide

  • Block non-specific binding with 5% normal goat serum prior to antibody incubation

The quantitative immunohistochemical analysis in human bladder tissue demonstrates that optimized antigen retrieval enables detection of differential DAXX expression between normal urothelium and urothelial carcinoma .

Why might I observe different DAXX staining patterns in different cell types or tissues?

Differential DAXX staining patterns may result from several biological and technical factors:

• Biological variations:

  • Expression levels vary across cell and tissue types

  • Post-translational modifications affect epitope accessibility

  • In normal urothelium, DAXX shows stronger staining in superficial cell layers compared to basal cells

  • In urothelial carcinoma, this gradient is altered with lower staining in cells adjacent to stroma

• Subcellular localization differences:

  • DAXX predominantly localizes to the nucleus but can translocate to the cytoplasm under stress conditions

  • Nuclear localization often appears as a speckled pattern corresponding to PML nuclear bodies

  • Cytoplasmic translocation may occur during apoptotic signaling

• Chromatin status:

  • DAXX functions as a chromatin remodeler, and its association with chromatin varies with chromatin condensation state

  • Nuclear staining intensity correlates with chromatin organization patterns

• Technical considerations:

  • Different fixation methods affect epitope preservation

  • Variations in antigen retrieval efficiency across tissues

  • Antibody clones recognize different epitopes with variable accessibility

In quantitative analyses of normal urothelium versus urothelial carcinoma, researchers found that 70% of normal urothelium nuclei were immunostained, whereas 90% of carcinoma nuclei were positive, with distinct differences in staining intensity and distribution .

How can I distinguish between specific and non-specific signals when using DAXX antibodies?

Distinguishing specific from non-specific signals requires systematic controls and analysis:

• Essential controls:

  • Negative controls: DAXX knockout cells or tissues show complete absence of specific signal

  • Antibody controls: Replace primary antibody with non-immune serum of the same species

  • Absorption controls: Pre-incubate antibody with immunizing peptide to block specific binding

• Signal characteristics for specific DAXX staining:

  • Subcellular localization: Predominantly nuclear with enrichment in nuclear speckles/PML bodies

  • Molecular weight: ~100-120 kDa band on Western blots despite calculated MW of 81 kDa

  • Signal ablation: Complete loss of signal in DAXX knockout cells

• Common non-specific patterns to watch for:

  • Uniform cytoplasmic staining without nuclear signal

  • Membrane-only staining

  • Signal persisting in knockout controls

  • Multiple bands on Western blot that don't disappear in knockout samples

• Validation in multiple applications:

  • Confirm findings across complementary techniques (e.g., WB, IF, IHC)

  • Consistency across different antibodies targeting distinct DAXX epitopes

Researchers have demonstrated specific DAXX detection using knockout validation in HeLa, HAP1, and HCT116 cell lines, showing complete absence of the ~100 kDa band in knockout samples .

How should I quantitatively analyze DAXX immunostaining in tissue samples?

Quantitative analysis of DAXX immunostaining provides valuable insights into expression patterns:

• Image acquisition considerations:

  • Use consistent exposure settings across samples

  • Capture multiple representative fields (≥5) per sample

  • Include calibration standards for intensity normalization

• Nuclear measurement parameters:

  • Nuclear area: Measure in square microns using calibrated software

  • Staining intensity: Quantify using gray level values (lower values indicate stronger staining)

  • Percentage of positive nuclei: Calculate proportion of stained versus total nuclei

• Compartment-specific analysis:

  • For stratified epithelia, separately analyze:

    • Basal layer (adjacent to stroma)

    • Intermediate cell layers

    • Superficial/luminal cell layer

• Statistical approaches:

  • Compare mean gray values between experimental groups

  • Analyze at least 40 nuclei per location for statistical robustness

  • Apply appropriate statistical tests (e.g., Student's t-test)

In a study comparing normal urothelium to urothelial carcinoma, researchers found:

• UC nuclei were 1.7× larger than normal urothelium nuclei (UC: 24.4±11.4 μm² vs. NU: 14.8±6.5 μm²)

• Mean gray level value in UC was lower than in NU by a factor of 0.94 (UC: 100±15 vs. NU: 106±15)

• 70% of normal urothelium nuclei were DAXX-positive versus 90% in urothelial carcinoma

How can DAXX antibodies be used to study chromatin remodeling and histone deposition?

DAXX functions as a histone chaperone in the ATRX:DAXX complex, making DAXX antibodies valuable tools for chromatin research:

• Chromatin immunoprecipitation (ChIP) applications:

  • Identify genomic regions where DAXX is recruited

  • Study DAXX association with histone H3.3-enriched regions

  • Investigate DAXX recruitment to pericentric and telomeric regions

• Co-immunoprecipitation studies:

  • Isolate DAXX-containing complexes to identify interaction partners

  • Study DAXX association with ATRX, histone H3.3, and PML bodies

  • Analyze how these interactions change under different conditions

• Fluorescence microscopy approaches:

  • Visualize DAXX colocalization with histone H3.3

  • Track DAXX recruitment to PML nuclear bodies

  • Study dynamics of DAXX-mediated histone deposition at specific genomic loci

• Functional studies:

  • Combine DAXX antibodies with DAXX knockdown/knockout

  • Analyze histone H3.3 deposition patterns in DAXX-deficient cells

  • Investigate changes in chromatin organization using DAXX antibodies as markers

DAXX has been shown to facilitate replication-independent deposition of histone H3.3 in pericentric DNA repeats and telomeres, and is required for the recruitment of histone H3.3:H4 dimers to PML nuclear bodies .

What is the role of DAXX in cancer biology and how can DAXX antibodies contribute to cancer research?

DAXX has emerging roles in cancer biology, making DAXX antibodies important tools in cancer research:

• DAXX alterations in cancer:

  • Somatic mutations in DAXX occur in alternative lengthening of telomeres (ALT) cancers

  • DAXX is frequently mutated in pancreatic neuroendocrine tumors

  • Altered DAXX expression serves as a marker of aggressiveness in urothelial carcinoma

• Research applications of DAXX antibodies in cancer:

  • Expression analysis: Quantify DAXX levels across tumor types and grades

  • Prognostic markers: Correlate DAXX expression patterns with clinical outcomes

  • Chromatin studies: Investigate how DAXX alterations affect chromatin organization in cancer cells

• Methodological approaches:

  • Tissue microarrays: Analyze DAXX expression across large tumor cohorts

  • Immunohistochemical scoring: Develop standardized scoring systems for DAXX expression

  • Multi-marker panels: Combine DAXX with other chromatin modifiers as diagnostic/prognostic tools

• Emerging research directions:

  • DAXX immunostaining in combination with markers of global DNA methylation and histone acetylation may serve as markers of tumor aggressiveness

  • Quantitative analysis of nuclear DAXX expression in preinvasive phases could help identify aggressive tumors

A quantitative immunohistochemical study demonstrated altered DAXX expression in urothelial carcinoma and its preinvasive phases compared to normal urothelium, suggesting potential roles as a marker of aggressiveness when combined with other epigenetic markers .

How can DAXX antibodies be used to investigate the relationship between DAXX and centromeric noncoding RNA transcription?

Recent research has revealed DAXX's role in regulating noncoding RNA transcription at centromeres:

• Research context:

  • DAXX promotes centromeric localization of ZFAT (Zinc finger protein with AT hook domain)

  • This localization regulates noncoding RNA transcription at centromeres

  • Dysregulation affects chromosome segregation and genomic stability

• Experimental approaches:

  • RNA interference: siRNA-mediated DAXX knockdown (72h transfection) followed by qRT-PCR to measure centromeric ncRNA levels

  • Immunofluorescence: Visualize DAXX and ZFAT colocalization at centromeres using confocal microscopy

  • Chromatin immunoprecipitation: Assess DAXX recruitment to centromeric regions

• Technical considerations:

  • Fix cells with 100% methanol for 20 min at -20°C for optimal centromeric protein detection

  • Permeabilize with 0.3% Triton X-100 to maintain nuclear architecture

  • Use laser-scanning confocal microscopy for precise colocalization analysis

• Methodological workflow:

  • Transfect cells with DAXX siRNA using Lipofectamine RNAiMAX

  • Extract RNA with TRIzol reagent and synthesize cDNA

  • Perform qPCR with specific primers for centromeric transcripts

  • Process cells in parallel for immunofluorescence to correlate DAXX levels with centromeric protein localization

This research area demonstrates how DAXX antibodies can help understand the complex relationship between chromatin proteins and noncoding RNA regulation at centromeres, with implications for chromosome segregation and genomic stability .

What methods can be used to study DAXX post-translational modifications using DAXX antibodies?

Post-translational modifications (PTMs) of DAXX regulate its functions and interactions:

• Key DAXX post-translational modifications:

  • Phosphorylation: Affects DAXX subcellular localization and protein interactions

  • SUMOylation: Influences DAXX association with PML nuclear bodies

  • Ubiquitination: Regulates DAXX protein stability and turnover

• Experimental approaches:

  • Phospho-specific antibodies: Detect specific phosphorylated residues on DAXX

  • 2D gel electrophoresis: Separate DAXX isoforms based on charge differences from PTMs

  • IP-Mass spectrometry: Immunoprecipitate DAXX and identify modifications by MS

  • Sequential immunoprecipitation: First IP with DAXX antibody, then probe with PTM-specific antibodies

• Methodological considerations:

  • Include phosphatase inhibitors in lysis buffers to preserve phosphorylation

  • Add SUMO protease inhibitors (e.g., N-ethylmaleimide) to maintain SUMOylation

  • Use proteasome inhibitors to prevent degradation of ubiquitinated forms

  • Compare DAXX migration patterns on Western blots before and after phosphatase treatment

• Advanced applications:

  • Proximity ligation assay (PLA): Detect specific modified forms of DAXX in situ

  • FRET analysis: Study how PTMs affect DAXX interactions with partners

  • Live cell imaging: Track how PTMs influence DAXX dynamics and localization

The observed higher molecular weight of DAXX in Western blots (100-120 kDa vs. calculated 81 kDa) likely reflects these numerous post-translational modifications , making careful sample preparation crucial for studying DAXX PTMs.