DAXX Antibody, FITC conjugated

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

DAXX (death domain-associated protein) is a ubiquitous protein implicated in various cellular processes including apoptosis, tumorigenesis, development, and transcriptional regulation. It has been shown to translocate from the nucleus to the cytoplasm under conditions of stress and activate the Jun N-terminal kinase (JNK) pathway . DAXX is particularly important in research because:

• It functions as a potent transcription repressor that binds to sumoylated transcription factors in the nucleus

• It associates with centromeres in G2 phase

• In the cytoplasm, it may function to regulate apoptosis

• Its subcellular localization and function are modulated by post-translational modifications including sumoylation, phosphorylation, and polyubiquitination

DAXX plays critical roles in multiple signaling pathways, making it a significant target for investigating cell death mechanisms, cancer progression, and cellular stress responses.

What are the primary applications for FITC-conjugated DAXX antibodies in research?

FITC-conjugated DAXX antibodies have several key applications in research settings:

• Immunofluorescence (IF): Direct visualization of DAXX localization within cells without requiring secondary antibodies

• Immunohistochemistry on paraffin-embedded tissues (IHC-P): Typically used at dilutions of 1:50-200

• Flow cytometry: Detection of intracellular DAXX protein expression in cell populations

• Immunocytochemistry (ICC): Examining DAXX expression patterns in cultured cells

The FITC conjugation (excitation = 495 nm, emission = 519 nm) enables direct fluorescent detection without additional staining steps, simplifying protocols and reducing background issues that can occur with secondary antibody approaches .

How should researchers optimize protocols for FITC-conjugated DAXX antibody staining?

Optimization of FITC-conjugated DAXX antibody staining protocols requires careful consideration of several parameters:

Fixation and Permeabilization:

• For immunofluorescence: 4% formaldehyde fixation for 10 minutes at room temperature followed by permeabilization with 0.1% PBS-Tween containing 1% BSA, 10% normal serum, and 0.3M glycine provides optimal results

• For flow cytometry: Methanol fixation may be preferred for nuclear protein detection

Antibody Concentration Optimization:
Different applications require different dilutions:

Antigen Retrieval:
For IHC applications, antigen retrieval methods significantly impact staining quality:

• TE buffer pH 9.0 is suggested as optimal

• Alternatively, citrate buffer pH 6.0 may be used with different results

Counterstaining Considerations:
When using FITC-conjugated antibodies, choose nuclear counterstains with non-overlapping emission spectra (e.g., DAPI) to avoid signal interference.

It is strongly recommended to titrate the antibody for each specific application and sample type to obtain optimal signal-to-noise ratios.

What controls should be included when working with FITC-conjugated DAXX antibodies?

Proper controls are essential for valid interpretation of DAXX antibody staining results:

Positive Controls:

• Y79 cells and HeLa cells have been validated for Western blot applications

• Human stomach tissue has been validated for IHC applications

Negative Controls:

• DAXX knockout cell lines (such as DAXX knockout HeLa cells) serve as ideal negative controls

• Isotype controls (FITC-conjugated non-specific rabbit IgG) should be used at the same concentration as the primary antibody

Specificity Controls:

• Blocking peptide competition assays can verify antibody specificity

• Dual staining with a different DAXX antibody (different clone or host) targeting another epitope can confirm specificity of observed signals

Subcellular Localization Controls:

• Nuclear markers (e.g., DAPI) should be used to verify the expected predominantly nuclear localization of DAXX under normal conditions

• Fas stimulation can be used as a positive control for DAXX cytoplasmic translocation experiments

How do researchers distinguish between nuclear and cytoplasmic DAXX localization patterns?

Distinguishing between nuclear and cytoplasmic DAXX localization is critical for understanding its functional state:

Methodological Approach:

• Use confocal microscopy with Z-stack imaging for accurate subcellular localization

• Co-stain with specific nuclear markers (DAPI, Hoechst) and plasma membrane markers (WGA)

• Perform nuclear/cytoplasmic fractionation followed by Western blot as a complementary biochemical approach

Interpretation Guidelines:

• Under normal conditions, DAXX predominantly localizes to the nucleus, particularly in PML nuclear bodies (PODs)

• Stress conditions can trigger translocation to the cytoplasm

• The nuclear/cytoplasmic ratio (NCR) of DAXX has prognostic significance in certain cancers

Research Findings:
Studies have demonstrated that cytoplasmic DAXX (cDAXX) and nuclear DAXX (nDAXX) have opposing biological functions in cancer contexts:

• In gastric cancer, cDAXX is associated with better survival while high nDAXX expression suggests poorer prognosis

• Upregulation of DAXX in the cytoplasm inhibits cell proliferation and promotes apoptosis

• Downregulation of DAXX in the nucleus produces opposite effects

Careful image analysis using nuclear boundary delineation and quantitative intensity measurements in both compartments is essential for accurate NCR calculations.

What is the relationship between DAXX expression patterns and disease states?

DAXX expression patterns have significant correlations with various disease states, particularly in cancer:

Gastric Cancer:

• Immunohistochemical studies of 323 gastric cancer tissues revealed differential prognostic implications based on DAXX localization

• High nuclear/cytoplasmic ratio correlates with poor prognosis

Pancreatic Neuroendocrine Tumors (PanNETs):

• Somatic mutations in DAXX are common in alternative lengthening of telomeres (ALT) cancers, including pancreatic neuroendocrine tumors

• Loss of DAXX nuclear expression by IHC has 85.2% sensitivity for detecting DAXX mutations, except when mutations occur in the last exon

Correlation with DAXX Mutations:

Implications for T-cell Function:

• Studies with transgenic mice expressing dominant-negative Daxx (Daxx-DN) show:

  • Protection of activated T cells from Fas-induced cell death

  • Increased proliferative T-cell responses

  • Increased tyrosine phosphorylation of LAT and ZAP70

These findings identify DAXX as a critical regulator of T-lymphocyte homeostasis through dual mechanisms: decreasing TCR-induced cell proliferation and promoting Fas-mediated cell death .

How can FITC-conjugated DAXX antibodies be used to study DAXX's role in apoptotic pathways?

FITC-conjugated DAXX antibodies provide valuable tools for investigating DAXX's complex role in apoptotic pathways:

Dual Immunofluorescence Studies:

• FITC-conjugated DAXX antibodies can be combined with red-fluorescent markers for apoptotic proteins (e.g., activated caspases, Fas receptor) to visualize co-localization during apoptosis

• This approach revealed that HPV16 E6 interacts with DAXX in C33A cells, affecting its subcellular distribution and apoptotic function

Live Cell Imaging Applications:

• Time-lapse fluorescence microscopy with FITC-DAXX antibody microinjection can track DAXX translocation in response to apoptotic stimuli

• Research has shown that DAXX binds specifically to the Fas death domain and enhances Fas-induced apoptosis while activating the JNK pathway

Functional Analysis Methodology:

• Induce apoptosis via death receptor activation (Fas ligand, TNF)

• Track DAXX translocation from nucleus to cytoplasm using FITC-DAXX antibodies

• Visualize interaction with ASK1 (apoptosis signal-regulating kinase 1) through proximity ligation assays

• Correlate with JNK activation and downstream apoptotic events

Research findings demonstrate that DAXX enhances CD95-mediated apoptosis in a FADD/procaspase-8-independent manner by directly binding to and activating ASK-1 .

What techniques can be used to study DAXX interactions with partner proteins using FITC-conjugated antibodies?

Analysis of DAXX protein interactions requires sophisticated methodological approaches:

Co-immunoprecipitation Combined with Immunofluorescence:

• Perform co-IP with anti-DAXX antibody followed by immunoblotting for interaction partners

• Complement with FITC-DAXX immunofluorescence to visualize subcellular localization of interactions

• This approach has demonstrated that DAXX interacts directly with SUMO-2/3, affecting its subcellular localization

Proximity Ligation Assay (PLA):

• Combining FITC-DAXX antibody with antibodies against suspected interaction partners

• PLA generates fluorescent signals only when proteins are in close proximity (<40 nm)

• Example findings using this approach:

  • Interaction between HPV16 E6 and DAXX was detected in the nuclear and cytoplasmic compartments in C33A cells

  • Blue fluorescence marked the nucleus, red fluorescence showed HPV16 E6 distribution in nucleus and cytoplasm, while green fluorescence of DAXX was primarily nuclear but redistributed to cytoplasm in cells expressing HPV16 E6

FRET-based Interaction Analysis:
When using FITC-conjugated DAXX antibodies as donors, partner proteins labeled with appropriate acceptor fluorophores can enable Förster Resonance Energy Transfer (FRET) analysis for detecting molecular interactions at nanometer resolution.

How do DAXX splice variants differ in their detection patterns with FITC-conjugated antibodies?

DAXX exists in multiple splice variants that exhibit distinct detection patterns:

Known DAXX Splice Variants:

• Standard DAXX (full-length)

• DAXX-β (deletion of nucleotides 19-170 of exon 6)

• DAXX-γ (additional alternative splice variant)

Detection Considerations:

• Antibody epitope location determines which variants will be detected

• Western blot may reveal multiple bands corresponding to different splice variants:

  • Calculated molecular weight of standard DAXX: 81 kDa

  • Observed molecular weights: 120 kDa and 70 kDa bands are commonly detected

Variant-Specific Analysis Approach:

The functional relevance of these splice variants remains an active area of research, with evidence suggesting they may have differential effects on apoptotic signaling pathways and transcriptional regulation.

What are common issues with FITC-conjugated DAXX antibody staining and how can they be resolved?

When working with FITC-conjugated DAXX antibodies, researchers frequently encounter several technical challenges:

High Background Signal:

• Cause: Insufficient blocking, excessive antibody concentration, or non-specific binding

• Solution: Increase blocking time (use 1% BSA/10% normal serum/0.3M glycine in 0.1% PBS-Tween) , optimize antibody dilution, and include 0.1-0.3% Triton X-100 in wash buffers

Weak or No Signal:

• Cause: Ineffective antigen retrieval, inadequate permeabilization, or epitope masking

• Solution: Compare antigen retrieval methods (TE buffer pH 9.0 versus citrate buffer pH 6.0) , increase permeabilization time, and ensure proper storage of antibody (maintain at -20°C with aliquoting for long-term stability)

Photobleaching:

• Cause: FITC is particularly susceptible to photobleaching

• Solution: Minimize exposure to light during staining, use anti-fade mounting media, and consider image acquisition optimization

Inconsistent Nuclear/Cytoplasmic Staining:

• Cause: Fixation artifacts, permeabilization issues, or biological variability

• Solution: Compare different fixation methods (4% PFA versus methanol), adjust permeabilization conditions, and consider positive controls with known DAXX localization patterns

Non-specific Binding:

• Cause: Insufficient washing, overfixation, or high antibody concentration

• Solution: Increase washing steps, optimize fixation time, and titrate antibody concentration for each experimental condition

How can researchers validate the specificity of FITC-conjugated DAXX antibodies?

Validation of FITC-conjugated DAXX antibody specificity is crucial for generating reliable research data:

Knockout/Knockdown Controls:

• Use DAXX knockout cell lines (e.g., DAXX knockout HeLa cells) as negative controls

• Compare with siRNA-mediated DAXX knockdown cells showing partial reduction in signal

Multiple Antibody Validation:

• Compare staining patterns with alternative DAXX antibody clones (e.g., E94 and 25C12 clones) targeting different epitopes

• All validated DAXX mutant PanNETs tested with both clone E94 and clone 25C12 showed concordant results

Blocking Peptide Competition:

• Pre-incubate antibody with excess immunogen peptide before staining

• Specific signals should be significantly reduced or eliminated

Western Blot Correlation:

• Perform parallel Western blot analysis to confirm the specificity of bands

• Expected molecular weights: calculated 81 kDa; observed 100-120 kDa and/or 70 kDa bands

• The discrepancy in molecular weight is attributed to post-translational modifications

Positive Control Tissues/Cells:

• Use validated positive controls:

  • Y79 cells and HeLa cells for WB applications

  • Human stomach tissue for IHC applications

  • Compare with previously published staining patterns in similar experimental systems

How is DAXX antibody immunohistochemistry being used as a surrogate marker for DAXX mutations?

DAXX immunohistochemistry (IHC) has emerged as a valuable screening tool for DAXX mutations, particularly in pancreatic neuroendocrine tumors (PanNETs):

Clinical Research Application:

• In a study of 27 PanNET cases with known DAXX mutations, 23 showed loss of DAXX expression by IHC while 4 retained expression

• This establishes DAXX IHC as having 85.2% sensitivity for detecting DAXX mutations

Correlation Between Mutation Location and IHC Detection:

• Mutations occurring outside the last exon typically result in loss of DAXX expression

• Mutations in the small ubiquitin-like modifier (SUMO)-Interaction motifs (SIMs) in the last exon may retain DAXX expression despite mutation

Optimization for Mutation Screening:

• Rabbit monoclonal antibody clone E94 (at 1:300 dilution) with high pH buffer (ER2) antigen retrieval provides optimal results

• 30-minute heat-based antigen retrieval ensures consistent staining patterns

This approach offers a cost-effective initial screening method for potential DAXX mutations before proceeding to more expensive and time-consuming sequencing techniques.

What is the significance of DAXX sumoylation in regulating its function and how can it be studied?

DAXX undergoes extensive post-translational modifications, with sumoylation playing a particularly crucial role in regulating its subcellular localization and function:

SUMO Modification of DAXX:

• DAXX expression significantly correlates with SUMO-2/3 in gastric cancer tissues (confirmed via GEPIA database analysis)

• Co-immunoprecipitation combined with immunofluorescence demonstrates direct interaction between DAXX and SUMO-2/3

Methodological Approaches:

• Bioinformatics Analysis: Using GEPIA to analyze correlations between DAXX and SUMO proteins

• Co-IP and Western Blot: To detect SUMO-modified DAXX forms

• Immunofluorescence with FITC-DAXX: To track subcellular localization changes following manipulation of SUMO-2/3 levels

• Sumoylation Site Mutagenesis: To determine critical residues for DAXX function

Research Findings:

• Downregulating SUMO-2/3 expression results in altered subcellular localization of DAXX

• Bioinformatics analysis suggests RanBP2 may function as a SUMO E3 ligase to promote nuclear-plasma transport by combining with RanGAP1

• This molecular network of cDAXX, nDAXX, and SUMO-2/3 regulates the subcellular localization of DAXX and thereby modulates its opposing biological effects

These findings have significant implications for understanding DAXX regulation in both normal and pathological conditions, potentially offering new therapeutic targets for diseases with dysregulated DAXX function.