DTX48 Antibody

What is DTX4 and what is its function in cellular processes?

DTX4 (Deltex 4) functions as a regulator of the Notch signaling pathway, which plays a critical role in cell-cell communications governing cell-fate determinations. At the molecular level, DTX4 acts as an E3 ubiquitin ligase protein, mediating 'Lys48'-linked polyubiquitination and promoting degradation of TBK1 (TANK-binding kinase 1). This targeting to TBK1 requires interaction with NLRP4 . Within the Deltex protein family, DTX4 contains specific structural domains that enable its functionality in ubiquitination processes and cellular regulatory mechanisms.

DTX4 is also known by several aliases:

• Deltex 4 homolog

• Deltex 4, E3 ubiquitin ligase

• Protein deltex-4

• RING finger protein 155

• RING-type E3 ubiquitin transferase DTX4

What antibody types are available for DTX4 detection, and what are their key differences?

Based on current research tools, there are multiple antibody options for DTX4 detection:

Each antibody demonstrates unique characteristics in sensitivity, specificity, and application versatility. Monoclonal antibodies typically offer higher specificity but might recognize limited epitopes, while polyclonal antibodies recognize multiple epitopes but may have increased cross-reactivity .

What cell types have been validated for DTX4 expression detection?

DTX4 expression has been successfully detected in several cell lines:

When performing immunofluorescence detection, specific staining was localized to the cytoplasm, suggesting the primary functional location of DTX4 under normal cellular conditions .

What are the optimal protocols for DTX4 detection by Western blot?

For optimal Western blot detection of DTX4, researchers should consider the following protocol parameters:

• Sample preparation:

  • Validated cell lines: K562 and SW13 cell lysates

  • Lysis buffer: Standard RIPA buffer with protease inhibitors

  • Loading amount: 20-50 μg total protein recommended

• Antibody conditions:

  • Primary antibody: 2 μg/mL of Mouse Anti-Human DTX1/DTX4 Monoclonal Antibody

  • Secondary antibody: HRP-conjugated Anti-Mouse IgG Secondary Antibody

  • Membrane type: PVDF membrane yields better results than nitrocellulose

  • Blocking solution: Standard 5% non-fat milk or BSA in TBST

• Detection parameters:

  • Expected molecular weight: Approximately 67 kDa

  • Conditions: Reducing conditions recommended

  • Buffer system: Immunoblot Buffer Group 1 has been validated

For quantitative Western blots, implementing biological replicates (n=3) and using housekeeping proteins such as GAPDH or β-actin as loading controls is essential for reliable data interpretation.

How can researchers effectively perform immunofluorescence staining for DTX4?

Successful immunofluorescence detection of DTX4 requires attention to several key methodological aspects:

• Cell preparation:

  • Validated models: K562 human chronic myelogenous leukemia cell line

  • Fixation method: Immersion fixation (4% paraformaldehyde, 15 minutes)

  • Permeabilization: 0.1% Triton X-100 for intracellular access

• Staining protocol:

  • Primary antibody concentration: 10 μg/mL of anti-DTX4 antibody

  • Incubation conditions: 3 hours at room temperature

  • Secondary antibody: NorthernLights 557-conjugated Anti-Mouse IgG

  • Nuclear counterstain: DAPI (blue)

• Analysis considerations:

  • Expected localization: Primarily cytoplasmic staining

  • Controls: Include secondary-only controls and isotype controls

  • Image acquisition: Z-stack imaging recommended to capture full signal distribution

For non-adherent cells like K562, specialized protocols such as cytospin preparation or poly-L-lysine coating may improve cell adherence during the staining procedure.

What strategies can researchers employ to validate DTX4 antibody specificity?

Antibody validation is crucial for generating reliable research data. For DTX4 antibodies, consider these validation approaches:

• Genetic validation methods:

  • siRNA/shRNA knockdown: Reduction in signal should correspond to knockdown efficiency

  • CRISPR-Cas9 knockout: Complete elimination of specific banding/staining

  • Overexpression: Enhanced signal in systems with tagged DTX4 constructs

• Protein-based validation:

  • Peptide competition assays: Pre-incubation with immunizing peptide should eliminate specific binding

  • Multiple antibody concordance: Different antibodies recognizing distinct epitopes should yield similar results

  • Immunoprecipitation followed by mass spectrometry: Confirms target identity

• Cross-reactivity assessment:

  • Testing across species: Evaluate performance in human, mouse, and rat samples

  • Homologous protein testing: Particularly important for distinguishing between DTX1 and DTX4, which share 97% sequence identity in certain regions

How can researchers study the interaction between DTX4 and the Notch signaling pathway?

Investigating DTX4's role in Notch signaling requires multifaceted experimental approaches:

• Protein-protein interaction studies:

  • Co-immunoprecipitation: Pull-down DTX4 and probe for Notch intracellular domain (NICD)

  • Proximity ligation assay: Visualize direct interactions between DTX4 and Notch components

  • FRET/BRET analysis: Real-time monitoring of dynamic interactions

• Functional pathway analysis:

  • Luciferase reporter assays: Using Notch-responsive elements (e.g., Hes1 promoter)

  • qRT-PCR for Notch target genes: Measure expression of Hes1, Hey1, etc. after DTX4 manipulation

  • Notch processing analysis: Western blot for NICD generation following DTX4 overexpression or knockdown

• Cellular phenotype assessment:

  • Differentiation assays in Notch-dependent cell types

  • Cell fate tracking in developmental models

  • Cell proliferation and survival analysis in cancer models where Notch signaling is active

What methodological approaches can effectively study DTX4's E3 ubiquitin ligase activity?

To investigate the ubiquitin ligase function of DTX4, researchers should consider these experimental strategies:

• In vitro ubiquitination assays:

  • Components: Purified DTX4, E1, E2 enzymes, ubiquitin, ATP, and potential substrates (e.g., TBK1)

  • Detection: Western blot for ubiquitin chains or substrate mobility shifts

  • Controls: Catalytically inactive DTX4 mutants (RING domain mutations)

• Cellular ubiquitination studies:

  • Co-expression: DTX4 with tagged ubiquitin and potential substrates

  • Immunoprecipitation: Pull-down substrate under denaturing conditions

  • Analysis: Western blot for 'Lys48'-linked polyubiquitin chains, which DTX4 is known to mediate

  • Proteasome inhibition: Treatment with MG132 to accumulate ubiquitinated proteins

• Substrate identification and validation:

  • Proteomics approach: Quantitative mass spectrometry comparing ubiquitinome in DTX4-depleted vs. control cells

  • Validation: Direct ubiquitination assays with identified substrates

  • Functional confirmation: Substrate stability and half-life measurement

How can researchers distinguish between DTX1 and DTX4 in experimental settings?

Given the high sequence similarity between DTX1 and DTX4 (97% identity in certain regions), distinguishing between these proteins requires careful experimental design:

• Molecular approaches:

  • Transcript-level analysis: Design primers targeting unique regions for qRT-PCR

  • Protein sequence targeting: Select antibodies raised against divergent regions

  • Epitope mapping: Characterize antibody binding sites using peptide arrays

• Functional discrimination:

  • Substrate specificity analysis: Identify differential ubiquitination targets

  • Interactome mapping: Determine unique binding partners for each protein

  • Knockout/knockdown phenotyping: Compare cellular outcomes of DTX1 vs. DTX4 depletion

• Technical verification methods:

  • Recombinant protein controls: Test antibody specificity using purified DTX1 and DTX4

  • Simultaneous detection: Use differently labeled antibodies to analyze co-expression

  • Mass spectrometry: Identify unique peptide fragments for definitive identification

What strategies can address inconsistent or high background signals when using DTX4 antibodies?

Researchers frequently encounter technical challenges with antibody-based detection. For DTX4 antibodies, consider these solutions:

• For high background in immunostaining:

  • Increase blocking stringency: Longer blocking time (2 hours) with 5-10% normal serum

  • Optimize antibody dilution: Test serial dilutions to find optimal concentration

  • Enhance washing steps: More frequent washes with higher detergent concentration (0.1-0.3% Tween-20)

  • Use alternative blocking agents: Casein, commercial blockers, or species-specific normal serum

• For multiple bands in Western blot:

  • Optimize sample preparation: Fresh lysates with complete protease inhibitor cocktails

  • Adjust denaturing conditions: Increase SDS concentration or boiling time

  • Implement gradient gels: Better separation of closely sized proteins

  • Consider post-translational modifications: Treatment with phosphatases or deglycosylation enzymes

• For poor signal-to-noise ratio in flow cytometry:

  • Optimize fixation/permeabilization: Test paraformaldehyde concentration (2-4%) and saponin (0.1-0.5%)

  • Improve blocking: Use Fc receptor blocking reagents for immune cells

  • Fluorophore selection: Choose brightest fluorophores for low-abundance targets

  • Include viability dye: Exclude dead cells which can bind antibodies non-specifically

How can researchers optimize DTX4 detection in challenging sample types?

Different sample types present unique challenges for antibody-based detection. For DTX4 research:

• Formalin-fixed paraffin-embedded (FFPE) tissues:

  • Antigen retrieval optimization: Compare heat-induced epitope retrieval methods (citrate vs. EDTA buffers)

  • Section thickness: 4-5 μm sections typically yield optimal results

  • Antibody incubation: Extended incubation (overnight at 4°C) may improve signal

  • Signal amplification: Consider tyramide signal amplification for low abundance detection

• Primary cell isolates:

  • Immediate fixation: Process samples quickly to preserve protein integrity

  • Gentle permeabilization: Titrate detergent concentration to maintain cellular structures

  • Blocking optimization: Extended blocking (1-2 hours) with serum matched to secondary antibody host

  • Antibody concentration: Higher concentrations may be needed compared to cell lines

• Tissue microarrays for high-throughput analysis:

  • Standardized protocol: Maintain consistent conditions across all samples

  • Positive and negative controls: Include on each array for quality control

  • Automated staining platforms: Reduce technical variability

  • Digital image analysis: Implement quantitative scoring for objective assessment

How can DTX4 antibodies be utilized in cancer research studies?

DTX4's role in signaling pathways makes it potentially relevant in cancer biology:

• Expression profiling applications:

  • Tumor tissue microarrays: Evaluate DTX4 expression across cancer types

  • Correlation analysis: Associate DTX4 levels with clinical outcomes

  • Single-cell analyses: Examine expression heterogeneity within tumors

• Functional investigations:

  • Drug response studies: Monitor DTX4 expression changes following treatment

  • Resistance mechanisms: Evaluate DTX4-mediated ubiquitination in therapy resistance

  • Combination approaches: Target DTX4-dependent pathways alongside standard treatments

• Methodological considerations:

  • Quantitative image analysis: Implement digital pathology tools for precise scoring

  • Multi-parameter flow cytometry: Co-staining with cancer stem cell markers

  • Spatial proteomics: Analyze DTX4 in relation to tumor microenvironment components

What role might DTX4 play in immune regulation and inflammatory conditions?

The ubiquitin ligase activity of DTX4 suggests potential immunoregulatory functions:

• Current understanding:

  • DTX4 mediates ubiquitination and degradation of TBK1, an essential component of innate immune signaling

  • This interaction requires NLRP4, suggesting involvement in inflammasome regulation

  • The Notch pathway, regulated by DTX proteins, plays crucial roles in immune cell development and function

• Investigative approaches:

  • Immune cell profiling: Analyze DTX4 expression across immune cell subsets

  • Stimulation experiments: Monitor DTX4 regulation during immune activation

  • Genetic models: Study immune phenotypes in DTX4-deficient systems

  • Cytokine profiling: Assess inflammatory mediator production following DTX4 manipulation

• Technical recommendations:

  • Flow cytometry panels: Include lineage markers alongside DTX4 staining

  • Ex vivo stimulation protocols: Standardize activation conditions

  • In vivo models: Consider tissue-specific conditional knockout approaches

How can advanced imaging techniques enhance DTX4 research?

Leveraging cutting-edge imaging methodologies can provide deeper insights into DTX4 biology:

• Super-resolution microscopy applications:

  • Structured illumination microscopy (SIM): 100-120 nm resolution for detailed subcellular localization

  • Stimulated emission depletion (STED): Visualize DTX4 interactions with specific cellular structures

  • Single-molecule localization microscopy: Precise spatial distribution at the nanoscale level

• Live-cell imaging approaches:

  • FRAP (Fluorescence Recovery After Photobleaching): Study DTX4 mobility and dynamics

  • FRET sensors: Monitor real-time protein-protein interactions with DTX4

  • Optogenetic tools: Control DTX4 activity with light-inducible systems

• Multiplexed imaging strategies:

  • Cyclic immunofluorescence: Sequential staining for 20+ proteins in the same sample

  • Mass cytometry imaging: Metal-labeled antibodies for highly multiplexed analysis

  • Spectral unmixing: Separate overlapping fluorophore signals for cleaner multi-color imaging