DAXX Antibody, Biotin conjugated

What is DAXX protein and why is it a significant research target?

Death-domain associated protein (DAXX) is a transcription repressor implicated in both physiological and pathological conditions. DAXX plays critical roles in apoptosis, tumorigenesis, development, and transcription regulation. Notably, DAXX overexpression is observed in diverse cancers, correlating with tumorigenesis, disease progression, and treatment resistance . DAXX has been shown to translocate from the nucleus to the cytoplasm under stress conditions, activating the Jun N-terminal kinase (JNK) pathway. Somatic mutations in DAXX have been identified in alternative lengthening of telomeres (ALT) cancers, including pancreatic neuroendocrine tumors . For researchers, DAXX represents an important target for understanding fundamental cellular processes and developing targeted cancer therapies.

What are the typical molecular weight characteristics of DAXX protein in experimental detection?

When working with DAXX antibodies, researchers should note that while the calculated molecular weight of DAXX is approximately 81 kDa (740 amino acids), the observed molecular weight in SDS-PAGE typically ranges between 100-130 kDa . Some antibodies detect DAXX at 120 kDa and 70 kDa . This discrepancy between calculated and observed molecular weights is likely due to post-translational modifications and should be considered when interpreting western blot results. Researchers should validate their specific antibody against appropriate positive controls to confirm detection at the expected molecular weight.

What applications are biotin-conjugated DAXX antibodies suitable for?

Based on current research data, biotin-conjugated DAXX antibodies are primarily optimized for:

When selecting a biotin-conjugated DAXX antibody, researchers should ensure it has been validated for their specific application of interest .

How does the amino acid targeting region affect DAXX antibody performance?

Different DAXX antibodies target distinct regions of the protein, which can significantly impact experimental outcomes:

When designing experiments, researchers should select antibodies targeting regions relevant to their biological question. For instance, if studying C-terminal interactions, an antibody targeting the AA 558-740 region would be more appropriate than one targeting the N-terminal region .

What are optimal storage conditions for maintaining biotin-conjugated DAXX antibody activity?

To preserve biotin-conjugated DAXX antibody function:

• Store at 4°C in the dark for short-term storage (up to 1 month)

• For long-term storage, maintain at -20°C

• Use storage buffers containing stabilizers such as 50% glycerol with PBS and 0.02% sodium azide

• Avoid repeated freeze-thaw cycles, which can lead to biotin degradation and compromised conjugate stability

• For small aliquots (20μL), adding 0.1% BSA can enhance stability during storage

• When stored properly, most biotin-conjugated antibodies remain stable for approximately one year after initial shipment

Improper storage can lead to decreased sensitivity, increased background signal, and potential false-negative results in experimental applications.

How should antigen retrieval be optimized when using biotin-conjugated DAXX antibodies for immunohistochemistry?

Antigen retrieval is critical for successful DAXX detection in fixed tissues. Evidence from multiple studies indicates:

For paraffin-embedded sections from human stomach tissue and human breast cancer tissue, TE buffer at pH 9.0 has been demonstrated to produce optimal staining with minimal background . When implementing antigen retrieval protocols, researchers should consider that:

• Retrieval time should be standardized (typically 15-20 minutes for heat-mediated methods)

• Cooling gradually to room temperature before antibody application enhances specific binding

• Different tissue types may require modified retrieval protocols

• When troubleshooting weak signals, extending retrieval time may improve detection

How can endogenous biotin interference be minimized when using biotin-conjugated DAXX antibodies?

Endogenous biotin can lead to false-positive results when using biotin-conjugated antibodies. To mitigate this issue:

• Implement a biotin blocking step using commercially available biotin blocking kits

• Pre-treat sections with avidin followed by biotin (sequential blocking)

• Use streptavidin-based detection systems with higher affinity for biotin than avidin-based systems

• For tissues with high endogenous biotin (kidney, liver), consider alternative conjugation methods

• Include appropriate negative controls processed identically but omitting the primary antibody

These measures are particularly important when working with biotin-conjugated DAXX antibodies in metabolically active tissues where endogenous biotin levels are elevated.

What are the recommended dilutions for biotin-conjugated DAXX antibodies across different applications?

Optimal dilution ranges vary by application and specific antibody:

Each application should be individually optimized, as the required concentration may vary depending on the expression level of DAXX in specific sample types .

How does biotin conjugation affect DAXX antibody binding and detection mechanisms?

Biotin conjugation fundamentally alters the detection strategy for DAXX antibodies through several mechanisms:

• Conjugation ratio: Most commercial biotin-conjugated DAXX antibodies contain 3-5 biotin molecules per antibody molecule. Higher ratios can lead to increased sensitivity but may compromise antigen binding if biotinylation occurs near the antigen-binding site .

• Binding kinetics: Biotin conjugation slightly increases the molecular weight of the antibody, potentially slowing tissue penetration in some applications. Extended incubation times may be necessary compared to unconjugated antibodies.

• Signal amplification: The biotin-streptavidin system allows for signal amplification, as multiple streptavidin-reporter molecules can bind to each biotinylated antibody. This is particularly advantageous when detecting low-abundance DAXX in certain cancer types .

What detection systems are most compatible with biotin-conjugated DAXX antibodies?

Selecting the appropriate detection system is crucial for maximizing sensitivity and specificity:

Research indicates that for immunohistochemical applications, Streptavidin-Biotin-Complex (SABC) with DAB chromogen provides optimal visualization of DAXX in paraffin-embedded sections of human intestinal cancer tissue and rat intestine tissue . For immunofluorescence applications involving co-localization studies, combining Cy3-conjugated anti-rabbit IgG and DyLight®488-conjugated anti-mouse IgG detection systems has been successfully employed to visualize DAXX alongside other proteins such as Tubulin Alpha in HeLa cells .

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

Thorough validation is essential to ensure experimental rigor:

• Knockout/knockdown validation: Use DAXX knockout cell lines as negative controls. Western blot analysis of wild-type versus DAXX knockout cells (such as knockout HAP1, A549, or HeLa cells) provides definitive evidence of antibody specificity .

• Peptide competition assays: Pre-incubation of the antibody with the immunizing peptide should abolish specific staining in all applications if the antibody is specific.

• Cross-validation with multiple antibodies: Compare staining patterns using antibodies targeting different epitopes of DAXX .

• Positive control tissues/cells: Include samples with known DAXX expression patterns (HeLa, Raji, Daudi, MOLT-4, Jurkat cells have been validated as positive controls for DAXX detection) .

• Molecular weight verification: Confirm detection at the expected molecular weight (100-130 kDa for DAXX) in western blot applications .

• Subcellular localization assessment: DAXX typically exhibits nuclear localization under normal conditions, with cytoplasmic translocation under stress conditions. Immunofluorescence studies should confirm this expected localization pattern .

What experimental controls are essential when using biotin-conjugated DAXX antibodies in complex tissue samples?

Implementing rigorous controls is critical for interpreting results with biotin-conjugated DAXX antibodies:

• Negative antibody control: Inclusion of isotype-matched, irrelevant biotinylated antibody to assess non-specific binding

• Endogenous biotin control: Samples processed with streptavidin-detection system but no primary antibody to evaluate endogenous biotin signal

• Tissue negative control: Include tissues known not to express DAXX or use DAXX knockout tissue sections

• Absorption control: Pre-absorption of biotin-conjugated DAXX antibody with recombinant DAXX protein should eliminate specific staining

• Gradient controls: Include samples with varying DAXX expression levels to demonstrate staining intensity correlation with expression

For immunohistochemical applications in tissues with high endogenous biotin (like kidney or liver), a biotin blocking step is especially critical to prevent false-positive results. When analyzing cancerous tissues, inclusion of adjacent normal tissue provides an internal reference for expression level comparison .

How can biotin-conjugated DAXX antibodies be effectively utilized in multiplex immunofluorescence studies?

Multiplex immunofluorescence enables simultaneous visualization of multiple targets, providing valuable context for DAXX localization and interactions:

• Sequential staining protocol:

  • Begin with biotin-conjugated DAXX antibody followed by streptavidin-fluorophore conjugate

  • Block remaining biotin binding sites with free biotin

  • Proceed with additional primary-secondary antibody pairs using different fluorophores

• Fluorophore selection considerations:

  • Choose fluorophores with minimal spectral overlap

  • When using biotin-conjugated DAXX antibody with streptavidin-Cy3 (red emission), pair with fluorophores in green/far-red channels

  • Successful multiplexing has been demonstrated using biotin-conjugated DAXX antibodies with Cy3 alongside DyLight®488-conjugated detection systems for other targets

• Optimization parameters:

  • Antibody concentration: May require reduction to 50-75% of single-staining protocols

  • Incubation times: Extended incubation (overnight at 4°C) improves penetration and specificity

  • Sequential vs. simultaneous: Sequential staining typically produces cleaner results but requires more time

• Validation approaches:

  • Single-staining controls for each antibody should be run in parallel

  • Absorption controls for each target protein should eliminate their respective signals

  • Signal isolation testing using secondary-only controls confirms minimal cross-reactivity

How are biotin-conjugated DAXX antibodies contributing to cancer research advances?

Recent studies utilizing biotin-conjugated DAXX antibodies have yielded significant insights into cancer biology:

• DAXX overexpression patterns: Immunohistochemical analyses using biotin-conjugated DAXX antibodies have revealed that DAXX overexpression is a common feature in diverse cancers, correlating with tumorigenesis, disease progression, and treatment resistance . This consistent finding across multiple studies establishes DAXX expression as a potential prognostic marker.

• Subcellular localization changes: Biotin-conjugated DAXX antibodies in immunofluorescence applications have demonstrated that nuclear-to-cytoplasmic translocation of DAXX occurs in response to cellular stress, potentially serving as an early marker of treatment response in cancer cells .

• Interaction mapping: Using proximity ligation assays (PLA) with biotin-conjugated DAXX antibodies, researchers have begun mapping protein-protein interactions involving DAXX in various cancer cell types, revealing potential therapeutic targets .

• Alternative lengthening of telomeres (ALT): Studies of pancreatic neuroendocrine tumors have employed biotin-conjugated DAXX antibodies to investigate the relationship between DAXX mutations and ALT, a telomerase-independent mechanism of telomere maintenance observed in approximately 10-15% of human cancers .

These findings collectively establish DAXX as a multifunctional protein with significant implications for cancer biology and potential therapeutic interventions.

What methodological advances are improving the reliability of biotin-conjugated antibody detection systems?

Recent technological developments have enhanced the utility of biotin-conjugated DAXX antibodies:

• Tyramide signal amplification (TSA): This technique amplifies detection signals by depositing multiple biotin molecules at the site of antibody binding, increasing sensitivity by 10-100 fold. This is particularly valuable for detecting low-abundance DAXX in certain tissue types.

• Quantum dot conjugates: Streptavidin-conjugated quantum dots offer superior photostability compared to conventional fluorophores, enabling extended imaging sessions and improved signal-to-noise ratios in DAXX localization studies.

• Biotin-conjugation chemistries: Advanced site-specific conjugation methods ensure that biotin molecules are attached away from the antigen-binding site, preserving antibody affinity while maintaining detection sensitivity .

• Multiplex imaging platforms: Mass cytometry (CyTOF) and imaging mass cytometry systems compatible with biotin-avidin detection systems enable highly multiplexed analyses of DAXX alongside dozens of other proteins, providing unprecedented insights into its functional contexts .

• Automated quantification: Digital pathology systems with sophisticated image analysis algorithms can quantify DAXX expression levels detected by biotinylated antibodies across whole tissue sections, enabling more objective and reproducible assessments.

These methodological advances are enabling more nuanced investigations of DAXX biology and its implications for disease mechanisms and therapeutic approaches.