DTX44 Antibody

What is DTX4 and what cellular functions does it regulate?

DTX4 (Deltex E3 Ubiquitin Ligase 4) is a regulator of Notch signaling, a pathway involved in cell-cell communications that governs a broad spectrum of cell-fate determinations. It functions as a ubiquitin ligase protein in vivo, mediating 'Lys-48'-linked polyubiquitination and promoting degradation of TBK1 (TANK-binding kinase 1). This targeting to TBK1 requires interaction with NLRP4. DTX4 is primarily localized in the cytoplasm .

What are the molecular characteristics of DTX4 protein that researchers should be aware of?

DTX4 has a predicted molecular weight of approximately 68 kDa, which is confirmed in Western blot analyses. The protein has multiple aliases including RING finger protein 155 (RNF155) and KIAA0937. At least two isoforms of DTX4 are known to exist, and both can be detected by certain antibodies. The human DTX4 protein is encoded by gene ID 23220, while the mouse homolog is encoded by gene ID 207521 .

What are the standard applications for DTX4 antibodies in research?

DTX4 antibodies are commonly used in several standard laboratory techniques:

• Western blot (WB) at 1-2 μg/ml concentration

• Immunohistochemistry (IHC) starting at 5 μg/ml

• Immunofluorescence (IF) starting at 20 μg/ml

• Flow cytometry (FCM) for detection of cellular expression patterns
  These applications allow researchers to detect endogenous levels of total DTX4 protein in various experimental systems .

How should researchers optimize Western blot protocols for DTX4 detection?

For optimal DTX4 detection by Western blot:

• Use cell lysates from appropriate cell lines (e.g., HeLa cells show detectable expression)

• Load adequate protein (typically 20-50 μg of total protein per lane)

• Perform SDS-PAGE under reducing conditions

• Use Immunoblot Buffer Group 1 or similar buffer systems

• Probe membranes with DTX4 antibody at 1-2 μg/ml concentration

• Use appropriate HRP-conjugated secondary antibodies (anti-rabbit for polyclonal antibodies)

• Expect to observe a specific band at approximately 68 kDa
  Reducing conditions are critical for proper DTX4 detection, as demonstrated in detection protocols for various cell lines .

What controls should be included when validating DTX4 antibody specificity?

To validate DTX4 antibody specificity, researchers should include:

• Positive control: Lysates from cell lines known to express DTX4 (e.g., HeLa cells)

• Negative control: Either lysates from cell lines with low/no DTX4 expression or samples treated with DTX4 siRNA

• Isotype control antibody to exclude non-specific binding

• Loading control (e.g., β-actin, GAPDH) to ensure equal protein loading

• Cross-reactivity testing against other DTX family members
  Most DTX4 antibodies are predicted not to cross-react with other DTX protein family members, but this should be experimentally validated for critical applications .

What are the optimal storage conditions for maintaining DTX4 antibody activity?

For maximum stability and retention of activity:

• Store unopened antibody at -20°C to -70°C for up to 12 months from receipt date

• After reconstitution, store at 2-8°C under sterile conditions for up to 1 month

• For longer storage after reconstitution, aliquot and store at -20°C to -70°C for up to 6 months

• Avoid repeated freeze-thaw cycles as they can denature antibodies and reduce activity

• Use a manual defrost freezer for storage

• When handling, keep antibodies on ice and minimize exposure to room temperature .

How can DTX4 antibodies be used to study Notch signaling pathways in different cell types?

For investigating DTX4's role in Notch signaling:

• Cellular localization studies: Use immunofluorescence with DTX4 antibodies (starting at 20 μg/ml) to visualize subcellular distribution, particularly during Notch activation. Co-stain with markers for Notch pathway components to assess colocalization.

• Protein interaction analysis: Employ co-immunoprecipitation with DTX4 antibodies to pull down protein complexes, followed by probing for Notch pathway components.

• Signaling dynamics: Stimulate cells with Notch ligands and track DTX4 expression, localization, and interaction changes using time-course experiments.

• Differential expression analysis: Compare DTX4 expression patterns across cell types with varying Notch pathway activity using flow cytometry with permeabilization.
  This approach provides insights into how DTX4 regulation contributes to different Notch-dependent cellular outcomes in development and disease contexts .

What methodological approaches can differentiate between DTX4 isoforms in experimental systems?

To differentiate between DTX4 isoforms:

• Targeted antibody selection: Use isoform-specific antibodies when available. Some antibodies detect both known isoforms while others may be isoform-specific.

• Western blot optimization: Use high-resolution SDS-PAGE (8-10% gels run for extended periods) to separate closely-sized isoforms.

• 2D gel electrophoresis: Separate isoforms based on both molecular weight and isoelectric point differences.

• RT-PCR analysis: Design primers specific to each isoform for transcript-level distinction.

• Mass spectrometry: Use proteomic approaches to identify unique peptides specific to each isoform.
  When interpreting results, researchers should be aware that at least two isoforms of DTX4 are known to exist, and antibody reactivity with each should be considered when planning experiments .

How can researchers study the functional relationship between DTX4 and TBK1 using antibody-based techniques?

To investigate DTX4-TBK1 interactions:

• Co-immunoprecipitation (Co-IP): Use DTX4 antibodies to pull down protein complexes, then probe for TBK1, or vice versa.

• Proximity ligation assay (PLA): Visualize DTX4-TBK1 interactions in situ using DTX4 and TBK1 primary antibodies with PLA probes.

• Ubiquitination assays: Immunoprecipitate TBK1 and probe for ubiquitin to assess DTX4's E3 ligase activity on TBK1.

• Degradation kinetics: Perform pulse-chase experiments with cycloheximide treatment to monitor TBK1 degradation rates in the presence or absence of DTX4.

• NLRP4 dependency studies: Use siRNA to knock down NLRP4 and assess how this affects DTX4-TBK1 interactions.
  These approaches help elucidate how DTX4 mediates 'Lys-48'-linked polyubiquitination and promotes degradation of TBK1, with the targeting requiring interaction with NLRP4 .

What are common causes of non-specific binding with DTX4 antibodies and how can they be addressed?

Common causes of non-specific binding and their solutions:

• Insufficient blocking: Increase blocking time (1-2 hours) and use 5% BSA or milk in TBS-T.

• Excessive antibody concentration: Titrate antibody concentrations, starting with lower concentrations (0.5-1 μg/ml) for Western blot.

• Insufficient washing: Increase number and duration of wash steps (5-6 washes, 5-10 minutes each).

• Sample overloading: Reduce protein load to 20-30 μg per lane.

• Cross-reactivity: Validate antibody specificity using knockout/knockdown controls.

• Buffer incompatibility: Ensure Immunoblot Buffer Group 1 or equivalent is used as recommended.
  For critical applications, include appropriate negative controls and consider pre-adsorption of antibody with recombinant DTX4 protein to reduce non-specific binding .

How should researchers interpret differences in DTX4 detection between tissue types and experimental models?

When interpreting variable DTX4 detection:

• Baseline expression differences: DTX4 expression varies naturally between tissues and cell types. Compare to literature-reported expression patterns.

• Isoform prevalence: Different tissues may express different DTX4 isoforms, affecting antibody recognition.

• Post-translational modifications: Phosphorylation, ubiquitination, or other modifications may mask epitopes in certain contexts.

• Technical factors: Tissue-specific extraction protocols may affect protein recovery and epitope preservation.

• Antibody validation: Confirm antibody performance in each specific tissue type using positive and negative controls.
  Compare multiple detection methods (e.g., Western blot, IHC, RT-PCR) to build a comprehensive understanding of true expression patterns versus technical artifacts .

What methodological challenges arise when comparing results from different anti-DTX4 antibody clones?

When comparing results from different antibody clones:

• Epitope differences: Different clones recognize distinct epitopes which may be differentially accessible depending on protein conformation or interaction state.

• Clone-specific sensitivity: Establish detection thresholds for each clone using standardized samples.

• Background patterns: Each clone may have unique non-specific binding profiles that should be characterized.

• Application optimization: A clone that works well for Western blot may not be optimal for IHC or IF.

• Data normalization: When comparing quantitative data, normalize to consistent internal controls.
  When publishing or presenting such comparative data, clearly specify antibody clone information (clone number, catalog number, manufacturer) and the specific experimental conditions used with each .

How do research applications of DTX4 antibodies compare with those for other DTX family members?

Comparative analysis of DTX family antibody applications:

What methodological differences should researchers consider when working with DUX4 versus DTX4 antibodies?

Key methodological differences between DUX4 and DTX4 antibody applications:

• Protein size detection: DUX4 is detected at approximately 55 kDa, while DTX4 is detected at approximately 68 kDa on Western blots.

• Expression systems: DUX4 is often studied in inducible expression systems due to its cytotoxicity, while DTX4 can be studied in endogenous systems.

• Cell models: DUX4 research often uses muscle cell models due to its role in FSHD, while DTX4 studies span diverse cell types.

• Epitope accessibility: DUX4 as a transcription factor may require nuclear extraction protocols for optimal detection.

• Toxic effects: DUX4 expression can lead to cell death, necessitating careful timing in experimental designs.
  When designing experiments, researchers should note that these proteins serve distinct biological functions despite their similar abbreviated names (DTX4 is a Notch regulator; DUX4 is a transcription factor), requiring different experimental approaches .

What complementary techniques should be used alongside DTX4 antibody-based methods to comprehensively study its function?

Complementary approaches to antibody-based DTX4 research:

• Genetic manipulation: CRISPR/Cas9 knockout or siRNA knockdown to assess loss-of-function phenotypes.

• Overexpression studies: Transfection with tagged DTX4 constructs to observe gain-of-function effects.

• Ubiquitination assays: In vitro ubiquitination assays to directly measure E3 ligase activity.

• Protein-protein interaction screens: Yeast two-hybrid or BioID proximity labeling to identify novel interaction partners.

• Transcriptomics: RNA-seq to identify genes regulated downstream of DTX4 activity.

• In vivo models: Conditional knockout mouse models to study tissue-specific DTX4 functions.

• Structure studies: X-ray crystallography or cryo-EM to analyze DTX4 protein structure.
  Integration of these approaches with antibody-based methods provides a more complete understanding of DTX4 function in cellular signaling networks and disease contexts .

How can DTX4 antibodies contribute to understanding its role in immune regulation and inflammatory diseases?

DTX4's function as a ubiquitin ligase that mediates degradation of TBK1 suggests it plays a regulatory role in immune signaling. Researchers can explore this using:

• Immune cell profiling: Analyze DTX4 expression across immune cell populations using flow cytometry with DTX4 antibodies.

• Cytokine stimulation studies: Monitor DTX4 expression and localization changes following treatment with inflammatory cytokines.

• Infection models: Assess DTX4 dynamics during viral or bacterial challenges in cellular models.

• TBK1-dependent pathway analysis: Use DTX4 antibodies to track how DTX4 expression/activity affects TBK1-dependent antiviral responses.

• NLRP4 interaction studies: Investigate how NLRP4-DTX4 interactions change during inflammatory responses.
  These approaches can reveal how DTX4 contributes to immune homeostasis and potentially identify intervention points for inflammatory disorders .

What methodological approaches can integrate DTX4 antibody data with systems biology to map its functional networks?

Integrating DTX4 antibody data with systems biology approaches:

• Multiplex immunoassays: Use DTX4 antibodies alongside antibodies against known partners and pathway components in high-throughput formats.

• Phospho-proteomics: Combine DTX4 immunoprecipitation with phospho-proteomics to identify signaling networks affected by DTX4 activity.

• Spatial proteomics: Use imaging mass cytometry with DTX4 antibodies to understand spatial organization of DTX4 networks.

• Mathematical modeling: Incorporate quantitative DTX4 expression/activity data into computational models of Notch signaling.

• Multi-omics integration: Correlate antibody-based DTX4 protein measurements with transcriptomic and metabolomic data.

• Network analysis: Map DTX4 into protein interaction networks using both experimental and predicted interaction data.
  This integrated approach helps position DTX4 within broader cellular signaling networks and reveals emergent properties not evident from isolated studies .

How might DTX4 antibodies be employed in developing novel therapeutic approaches targeting Notch signaling pathways?

Applications in therapeutic development:

• Target validation: Use DTX4 antibodies to confirm target expression in disease tissues and validate knockdown efficiency in preclinical models.

• Pharmacodynamic biomarkers: Develop assays using DTX4 antibodies to monitor drug effects on Notch pathway activity.

• Patient stratification: Assess DTX4 expression patterns in patient samples to identify potential responders to Notch-targeting therapies.

• Antibody-drug conjugates: Explore the potential for DTX4-targeted antibody-drug conjugates in diseases with DTX4 overexpression.

• Mechanism of action studies: Use DTX4 antibodies to elucidate how experimental drugs affect DTX4 expression, localization, and function.

• Resistance mechanisms: Investigate changes in DTX4 expression or activity in models of resistance to Notch pathway inhibitors.
  These applications contribute to both basic understanding of Notch pathway regulation and translational opportunities in diseases where Notch signaling is dysregulated .