DTX26 Antibody

What is DTX2 and what are its primary functions in cellular processes?

DTX2 (Deltex homolog 2) functions as an E3 ubiquitin ligase and serves as a regulator of Notch signaling, a pathway involved in cell-cell communications that governs cell-fate determinations. It acts as both a positive and negative regulator of Notch, depending on developmental and cellular contexts. DTX2 mediates the antineural activity of Notch, possibly by inhibiting transcriptional activation mediated by MATCH1 . Its ubiquitin ligase activity suggests it may regulate the Notch pathway through protein ubiquitination mechanisms .
Recent research has revealed DTX2's oncogenic role in glioma development. It interacts with helicase-like transcription factor (HLTF) and induces its ubiquitination, thereby promoting glioma cell proliferation and migration while inhibiting apoptosis .

What experimental applications are DTX2 antibodies suitable for?

Most commercially available DTX2 antibodies have been validated for multiple applications with varying degrees of effectiveness:

How should I design experiments to investigate DTX2's role in the ubiquitination pathway?

When investigating DTX2's role in ubiquitination pathways, a comprehensive experimental approach should include:

• Protein interaction studies:

  • Co-immunoprecipitation assays to identify binding partners (as demonstrated in glioma cell studies with HLTF)

  • Confocal microscopy to visualize subcellular co-localization (DTX2 and HLTF signals were shown to co-locate in the nucleus)

• Ubiquitination assays:

  • In vitro ubiquitination assays to confirm DTX2's ability to downregulate target protein levels by increasing ubiquitination

  • Western blotting to detect changes in target protein expression levels following DTX2 knockdown or overexpression

• Functional validation:

  • Lentivirus-mediated gene knockdown and overexpression to determine the effects of DTX2 on cellular processes

  • Cell viability, colony formation, and migration assays to assess phenotypic consequences

  • Flow cytometry to analyze cell cycle distribution and apoptosis

• In vivo verification:

  • Subcutaneous xenograft models to confirm effects observed in vitro
    Include appropriate controls and consider the temporal dynamics of ubiquitination processes when designing these experiments.

What are the optimal sample preparation protocols for DTX2 antibody applications in different tissue types?

Sample preparation protocols vary by application and tissue type:
For Western Blot:

• Extract proteins using RIPA buffer supplemented with protease inhibitors

• For cellular fractionation studies, use specialized nuclear/cytoplasmic extraction kits as DTX2 has been shown to localize primarily in the nucleus

• Typically load 20-50 μg of total protein per lane

• Look for bands at approximately 67 kDa (calculated molecular weight)
  For Immunohistochemistry:

• For paraffin-embedded tissues: Perform antigen retrieval using citrate buffer (pH 6.0) or EDTA buffer (pH 9.0) depending on the specific antibody

• Blocking should be performed with 1-5% BSA or normal serum from the same species as the secondary antibody

• Typical dilutions range from 1:50-1:200

• DTX2 expression has been successfully detected in glioma tissues of varying grades
  For Immunofluorescence:

• Fix cells in 4% paraformaldehyde

• Permeabilize using 0.5% Triton X-100

• Co-stain nuclei with Hoechst 33342 or DAPI

• Visualize under confocal laser scanning microscope

Why might DTX2 antibodies show inconsistent results in glioma tissue sections?

Inconsistent results in glioma tissue sections can be attributed to several factors:

• Tissue heterogeneity:

  • Glioma tissues show variable DTX2 expression patterns based on grade (G1/G2 vs. G3/G4)

  • Expression varies significantly between individual patient samples, as confirmed by IHC staining

• Technical variables:

  • Fixation time and conditions significantly impact epitope preservation

  • Antigen retrieval methods may need optimization for specific tissue types

  • Different antibody clones target different epitopes, leading to variability in staining patterns

• Antibody specificity issues:

  • Cross-reactivity with other deltex family members (DTX1, DTX3, DTX4)

  • Background signals from endogenous biotin or peroxidase activity
    To mitigate these issues:

• Include both positive and negative controls in each experiment

• Use validated antibodies with proven specificity

• Optimize antigen retrieval and staining protocols for each tissue type

• Consider using multiple antibodies targeting different epitopes to confirm results

• Quantify DTX2 expression levels using standard scoring systems (e.g., H-score or percentage of positive cells)

How can I validate the specificity of a DTX2 antibody for my research?

To validate DTX2 antibody specificity, implement a multi-faceted approach:

• Genetic validation:

  • Use DTX2 knockdown (shRNA) and overexpression systems as positive and negative controls

  • Compare antibody signal between wild-type and genetically modified samples

• Western blot validation:

  • Confirm single band at expected molecular weight (67 kDa)

  • Preabsorption test with immunizing peptide should abolish specific signals

• Multiple antibody approach:

  • Use antibodies targeting different epitopes of DTX2 (N-terminal vs. C-terminal)

  • Comparable results with different antibodies increase confidence in specificity

• Cross-species reactivity:

  • Test antibody on samples from multiple species if working with non-human models

  • Many DTX2 antibodies have predicted reactivity with mouse (74%) and rat (75%) orthologs

• Immunoprecipitation followed by mass spectrometry:

  • Identify all proteins pulled down by the antibody

  • Confirm DTX2 as the predominant protein in the precipitate

• Immunofluorescence co-localization:

  • Use dual staining with antibodies against known interaction partners like HLTF

  • Co-localization patterns should match expected subcellular distribution

How can I investigate the differential roles of DTX2 in normal tissue versus cancer progression?

Investigating DTX2's differential roles requires a comprehensive approach:

What are the methodological considerations for studying DTX2's interactions with the HLTF protein complex?

Studying DTX2-HLTF interactions requires specialized approaches:

• Protein-protein interaction verification:

  • Co-immunoprecipitation assays using whole-cell lysates of glioma cells expressing Flag-tagged DTX2

  • Reciprocal co-IPs pulling down either DTX2 or HLTF to confirm bidirectional interaction

  • Proximity ligation assays (PLA) to visualize interactions in situ

• Domain mapping:

  • Generate truncation mutants of both DTX2 and HLTF to identify interaction domains

  • Site-directed mutagenesis of key residues to pinpoint critical interaction sites

  • Yeast two-hybrid or mammalian two-hybrid systems to validate direct interactions

• Functional relationship studies:

  • Monitor HLTF protein levels following DTX2 knockdown or overexpression

  • In vitro ubiquitination assays to confirm DTX2's role in HLTF ubiquitination

  • Proteasome inhibition studies to determine if DTX2-mediated HLTF reduction occurs through proteasomal degradation

• Subcellular localization:

  • Immunofluorescence assays to visualize co-localization in the nucleus

  • Live-cell imaging with fluorescently tagged proteins to monitor dynamic interactions

  • FRET (Förster Resonance Energy Transfer) or BRET (Bioluminescence Resonance Energy Transfer) to measure proximity in living cells

• Structural studies:

  • Crystallography or cryo-EM to determine the structural basis of interaction

  • Hydrogen-deuterium exchange mass spectrometry to map interaction interfaces

How should I interpret contradictory findings between DTX2 expression levels and clinical outcomes across different cancer types?

When faced with contradictory findings regarding DTX2 expression and clinical outcomes:

What are the key considerations when analyzing DTX2's role in E3 ubiquitin ligase activity in different experimental systems?

When analyzing DTX2's E3 ubiquitin ligase activity:

• System-specific considerations:

  • Cell-free vs. cellular systems have different requirements for detecting ubiquitination

  • Primary cells vs. cell lines may exhibit different basal ubiquitination patterns

  • Consider the impact of cell cycle phase and cellular stress on ubiquitination dynamics

• Technical validation:

  • Include positive controls (known E3 ligases) and negative controls (catalytically inactive mutants)

  • Use multiple ubiquitination detection methods (western blot, ELISA, mass spectrometry)

  • Validate findings with both tagged and endogenous proteins

• Specificity determination:

  • Distinguish between mono-, multi-, and poly-ubiquitination

  • Identify ubiquitination sites using mass spectrometry

  • Determine ubiquitin chain topology (K48 vs. K63 linkages) which influences target protein fate

• Physiological relevance:

  • Correlate in vitro ubiquitination with protein stability in cells

  • Use proteasome inhibitors to confirm degradation-dependent effects

  • Perform cycloheximide chase assays to measure target protein half-life

• Network analysis:

  • Map DTX2 interactions within the broader ubiquitin-proteasome system

  • Identify potential co-factors or adaptors that enhance substrate specificity

  • Consider crosstalk with other post-translational modifications

How can bispecific antibody technology be applied to target DTX2 in combination with other cancer-associated proteins?

Bispecific antibody (bsAb) technology offers promising applications for targeting DTX2 in combination with other proteins:

• Design considerations for anti-DTX2 bsAbs:

  • Select appropriate scaffold formats based on intended mechanism of action

  • Common formats include IgG-like structures with symmetric or asymmetric designs

  • Consider single-chain Fab (scFab) domains to avoid HC:LC mispairing issues

• Target selection strategies:

  • Combine DTX2 targeting with tumor-specific antigens for enhanced selectivity

  • Target DTX2 alongside its interaction partners like HLTF for synergistic effects

  • Consider immune-engaging bsAbs targeting DTX2 and CD3 to recruit T cells

• Optimization approaches:

  • Engineer optimal binding affinities for each target

  • Address developability challenges through careful design to ensure favorable biophysical properties

  • Screen for candidates with minimal self-association and aggregation tendencies

• Potential advantages:

  • Simultaneous blocking of complementary oncogenic pathways

  • Improved tumor selectivity by requiring co-expression of both targets

  • Potential to overcome resistance mechanisms associated with single-target therapies

• Validation strategies:

  • Verify dual binding capabilities using surface plasmon resonance or bio-layer interferometry

  • Confirm functional effects in cellular assays relevant to known DTX2 functions

  • Evaluate efficacy in patient-derived xenograft models

What cutting-edge techniques are emerging for studying the dynamic interaction between DTX2 and chromatin remodeling complexes?

Several cutting-edge techniques can illuminate DTX2's interactions with chromatin remodeling complexes:

• Advanced imaging approaches:

  • Super-resolution microscopy (STORM, PALM) to visualize DTX2-chromatin interactions beyond diffraction limits

  • Live-cell single-molecule tracking to monitor dynamic associations with chromatin

  • Lattice light-sheet microscopy for long-term observation with minimal phototoxicity

• Chromatin interaction mapping:

  • ChIP-seq to identify DTX2-associated genomic regions

  • CUT&RUN or CUT&Tag for higher resolution mapping with lower background

  • HiChIP to connect DTX2 binding with 3D chromatin organization

• Protein-DNA proximity analysis:

  • APEX2-mediated proximity labeling to identify proteins near DTX2 at chromatin

  • Genome-wide DNA adenine methyltransferase identification (DamID) to map DTX2 binding without crosslinking

  • Chromatin immunoprecipitation with selective isolation of chromatin-associated proteins (ChIP-SICAP)

• Functional genomics integration:

  • CRISPR screening focused on chromatin remodelers to identify synthetic interactions with DTX2

  • CRISPRi/CRISPRa to modulate DTX2 or remodeler expression in specific genomic contexts

  • Multi-omics integration (RNA-seq, ATAC-seq, ChIP-seq) following DTX2 modulation

• Biochemical reconstitution:

  • In vitro reconstitution of DTX2-containing complexes on defined chromatin templates

  • Single-molecule assays to monitor real-time effects on nucleosome remodeling

  • Mass spectrometry-based approaches to identify post-translational modifications regulating these interactions This comprehensive FAQ collection provides researchers with methodological insights, experimental approaches, and analytical frameworks for investigating DTX2 antibodies in academic research contexts.