DUF4 Antibody

What is DUX4 and why is it significant in research?

DUX4 (Double Homeobox 4) is a transcription factor encoded by the DUX4 gene in humans. It has gained significant research attention as a candidate disease gene for facioscapulohumeral dystrophy (FSHD), one of the most common muscular dystrophies characterized by progressive skeletal muscle degeneration . More recently, DUX4 expression has been identified as a common feature in metastatic tumors, with approximately 10-50% of advanced bladder, breast, kidney, prostate, and skin cancers showing DUX4 expression, suggesting its role in immune evasion and immunotherapy failure in cancer . The protein has a reported molecular mass of 44.9 kilodaltons .

What types of DUX4 antibodies are available for research applications?

Researchers have developed multiple monoclonal antibodies targeting different regions of the DUX4 protein. Specifically, five monoclonal antibodies have been created that target the N- and C-termini of human DUX4 . Commercial suppliers currently offer at least 266 DUX4 antibodies across 23 different suppliers . These include mouse monoclonal antibodies like the P4H2 clone that specifically targets the C-terminus of human DUX4 (corresponding to Swiss-Prot: Q9UBX2) . Both conjugated and unconjugated forms are available depending on the intended application.

What are the validated applications for DUX4 antibodies?

DUX4 antibodies have been validated for multiple experimental techniques including:

• Western Blot (recommended concentration: 0.5-2.0 μg/ml)

• Simple Western (dilution 1:10)

• ELISA

• Immunohistochemistry (dilution 1:100-1:200)

• Immunocytochemistry/Immunofluorescence (dilution 1:100-1:200)

• Immunohistochemistry-Paraffin (dilution 1:100-1:200)

• Immunohistochemistry-Frozen (dilution 1:100-1:200)
  Immunofluorescence analysis demonstrates strong nuclear staining in cells expressing DUX4, consistent with its function as a transcription factor that binds to DNA sequences via its homeodomains to regulate target gene expression .

How should DUX4 antibodies be stored and handled for optimal performance?

For short-term storage, DUX4 antibodies should be kept at 4°C. For long-term storage, it is recommended to aliquot the antibody and store at -20°C to avoid repeated freeze-thaw cycles that can degrade antibody performance . Most commercial DUX4 antibodies are supplied in PBS buffer, and proper handling includes minimizing exposure to light and avoiding contamination. When working with these antibodies, researchers should follow supplier-specific instructions regarding reconstitution if provided in lyophilized form.

What controls should be included when validating DUX4 antibody specificity?

When validating the specificity of DUX4 antibodies, researchers should include:

• Positive controls: Cells transfected with DUX4 expression constructs

• Negative controls: Non-transfected neighboring cells that do not express DUX4

• Secondary antibody-only controls: To assess background staining

• Loading controls: For Western blot applications, such as anti-α-tubulin antibody

• Multiple antibody validation: Using different antibodies targeting distinct epitopes of DUX4 for confirmation
  Published studies have demonstrated that proper validation includes confirming nuclear localization in a punctate, granular pattern when using immunofluorescence techniques .

What is the recommended protocol for immunofluorescence detection of DUX4?

For optimal immunofluorescence detection of DUX4:

• Fix cells using appropriate fixative (paraformaldehyde is commonly used)

• Permeabilize cells to allow antibody access to nuclear DUX4

• Block with suitable blocking buffer to reduce non-specific binding

• Incubate with primary DUX4 antibody at recommended dilution (typically 1:100-1:200)

• Wash thoroughly to remove unbound primary antibody

• Incubate with fluorophore-conjugated secondary antibody

• Counterstain nuclei with DAPI or similar nuclear stain

• Mount and visualize using fluorescence microscopy
  Research has shown that cells expressing DUX4 display distinct nuclear staining not seen in neighboring cells that do not express DUX4. A punctate, granular pattern may indicate chromatin condensation and possible pre-apoptotic state .

How can DUX4 antibodies be utilized to study FSHD pathogenesis?

For studying FSHD pathogenesis using DUX4 antibodies, researchers should consider:

• Detecting native DUX4 expression in muscle biopsies from FSHD patients versus controls

• Analyzing the temporal dynamics of DUX4 expression in differentiated muscle cells

• Investigating alternatively spliced DUX4 isoforms using antibodies targeting different regions

• Performing co-immunoprecipitation experiments to identify DUX4 protein interaction partners

• Utilizing chromatin immunoprecipitation (ChIP) to identify DUX4 binding sites on genomic DNA
  Research has demonstrated that alternatively spliced transcripts from DUX4 exist in both FSHD and wild-type muscle cells, including the canonical full-length DUX4 and shorter splice forms that remove more than two-thirds of the coding region . Multiple antibody labeling experiments and combination methods such as immunoprecipitation followed by Western blot can be performed with anti-DUX4 antibodies produced from different species .

What are the considerations for detecting low levels of endogenous DUX4 expression?

Detecting low levels of endogenous DUX4 expression presents significant challenges that can be addressed through:

• Using highly sensitive detection methods such as immunoprecipitation followed by Western blot

• Employing signal amplification techniques like tyramide signal amplification for immunohistochemistry

• Utilizing more sensitive secondary detection systems (e.g., polymeric detection systems)

• Optimizing antibody concentration through careful titration experiments

• Considering cell fractionation to concentrate nuclear proteins before detection

• Using multiple antibodies targeting different epitopes to confirm weak signals
  Full-length DUX4 expression has been associated with cellular toxicity, making detection challenging in living cell systems. Additionally, the expression of different DUX4 isoforms may require isoform-specific antibodies for accurate detection .

How can DUX4 antibodies be employed in cancer research?

Recent findings demonstrating DUX4 expression in metastatic tumors suggest several applications for DUX4 antibodies in cancer research:

• Screening tissue microarrays from various cancer types to quantify DUX4 expression prevalence

• Correlating DUX4 expression with immune cell infiltration markers

• Exploring the relationship between DUX4 expression and immunotherapy response

• Investigating DUX4 as a potential biomarker for cancer progression and treatment resistance

• Studying the mechanisms by which DUX4 may contribute to immune evasion in cancer
  Research has shown that DUX4 expression is a common feature of metastatic tumors, with approximately 10-50% of advanced bladder, breast, kidney, prostate, and skin cancers expressing DUX4. This expression has been associated with features of immune evasion and may impact clinical outcomes in patients with advanced urothelial cancer .

What are common challenges when using DUX4 antibodies and how can they be addressed?

Common challenges and their solutions include:

How can antibody selection strategies impact experimental outcomes in DUX4 research?

The selection of appropriate antibodies significantly impacts experimental outcomes in DUX4 research:

• Multiple antibody targets may generate a huge computational cost for algorithms searching for the best model for the data

• For more than 5 antibody targets, brute-force approaches become computationally infeasible

• Data analysis strategies typically involve an antibody or feature selection stage followed by a predictive stage

• Parametric strategies can be employed for antibody selection, combining transformed and dichotomized antibody data

• Simpler strategies include initially dichotomizing antibody data using optimal cut-off points based on maximizing chi-square test statistics

• The positive correlation among different antibodies (average Spearman's correlation coefficient = 0.312) should be considered when interpreting results from multiple antibodies

What considerations should be made when selecting between monoclonal and polyclonal DUX4 antibodies?

When choosing between monoclonal and polyclonal DUX4 antibodies:

How might emerging antibody technologies enhance DUX4 detection and characterization?

Emerging antibody technologies poised to advance DUX4 research include:

• Single-domain antibodies (nanobodies) for enhanced tissue penetration and epitope accessibility

• Recombinant antibody fragments with improved specificity

• Proximity ligation assays for detecting protein-protein interactions involving DUX4

• Mass cytometry (CyTOF) using metal-conjugated antibodies for single-cell analysis

• Super-resolution microscopy compatible antibody conjugates for detailed spatial localization studies

• Multiplexed antibody-based imaging to simultaneously detect DUX4 and downstream targets
  These technologies could significantly enhance sensitivity and specificity for detecting low levels of endogenous DUX4 expression, which remains a significant challenge in FSHD research.

What research gaps exist in our understanding of DUX4 biology that could be addressed with improved antibodies?

Several key knowledge gaps could be addressed with improved DUX4 antibodies:

• The spatial and temporal expression patterns of DUX4 during normal development

• The prevalence and function of DUX4 isoforms in healthy tissues

• The mechanisms by which DUX4 contributes to immune evasion in cancers

• The post-translational modifications of DUX4 and their functional significance

• The protein interaction network of DUX4 in different cellular contexts

• The relationship between DUX4 expression levels and disease severity in FSHD Improved antibodies with higher sensitivity and specificity would enable more detailed investigations into these aspects of DUX4 biology and pathology.