DTX27 Antibody

What is the DTX2 protein and what biological pathways is it involved in?

DTX2 functions as an E3 ubiquitin ligase and plays a critical role in the Notch signaling pathway . This protein is crucial for cell-cell communications that regulate a broad spectrum of cell-fate determinations . DTX2 can act as both a positive and negative regulator of Notch signaling, depending on the developmental and cellular context . The protein mediates the antineural activity of Notch, possibly by inhibiting transcriptional activation mediated by MATCH1 . As an E3 ubiquitin ligase, DTX2 likely regulates protein degradation through the ubiquitin-proteasome pathway, suggesting its importance in controlling protein turnover in Notch signaling .

What are the primary applications for DTX2 antibodies in research?

DTX2 antibodies are optimized for multiple research applications, with Western blotting, immunohistochemistry-paraffin (IHC-P), and ELISA being the most common techniques . For Western blot applications, the recommended dilution range is typically 1:100 to 1:500, while for IHC-P applications, researchers should use dilutions between 1:50 and 1:200 . These antibodies enable researchers to detect and analyze DTX2 expression across various cell types, providing essential insights into its function and potential as a therapeutic target in diseases where Notch signaling is dysregulated . When designing experiments, researchers should confirm the reactivity of their chosen antibody with the species of interest, as some DTX2 antibodies show cross-reactivity with human, mouse, and rat samples .

What is the cellular localization of DTX2 and how does this affect experimental design?

DTX2 protein has been localized to both the cytoplasm and nucleus . This dual localization is significant when designing experiments, as researchers need to consider appropriate cell fractionation techniques when studying DTX2 in specific cellular compartments. For immunofluorescence studies, proper permeabilization protocols are crucial to ensure antibody accessibility to both compartments. When analyzing DTX2 function, researchers should consider that its activity may differ between these cellular locations, potentially relating to distinct roles in signaling or protein degradation pathways. The nuclear presence suggests possible involvement in transcriptional regulation, while cytoplasmic localization aligns with its E3 ligase activity in protein processing.

How should researchers validate the specificity of a DTX2 antibody?

Antibody validation is critical for ensuring experimental reliability. For DTX2 antibodies, a multi-faceted validation approach is recommended:

• Western blot analysis confirming a single band at the expected molecular weight of 67kDa

• Comparison with positive control samples (e.g., A-549 cell line)

• Employing knockout or knockdown controls where DTX2 is absent or reduced

• Peptide competition assays using the immunogen sequence

• Cross-validation using multiple antibodies targeting different epitopes of DTX2

Recent advances in antibody validation include biophysics-informed computational modeling, which can help predict antibody specificity profiles and identify potential cross-reactivity . This approach can be particularly valuable when working with closely related protein family members.

What methodological approaches are recommended for studying DTX2's E3 ubiquitin ligase activity?

To investigate DTX2's E3 ubiquitin ligase function, researchers should implement several specialized techniques:

• In vitro ubiquitination assays: Set up reactions containing purified DTX2, E1 and E2 enzymes, ubiquitin (preferably tagged), ATP, and potential substrate proteins. Analyze ubiquitination by Western blotting.

• Substrate identification: Employ immunoprecipitation with DTX2 antibodies followed by mass spectrometry to identify interacting proteins that may be substrates.

• Mutational analysis: Generate DTX2 variants with mutations in the RING domain (critical for E3 ligase activity) to create catalytically inactive controls.

• Ubiquitin chain analysis: Use antibodies specific for different ubiquitin linkages (K48, K63) to determine the type of chains formed by DTX2, which indicates the likely fate of the substrate.

• Proteasome inhibition studies: Treat cells with proteasome inhibitors to determine if DTX2-mediated ubiquitination leads to proteasomal degradation or other outcomes.

When conducting these experiments, researchers should consider that DTX2 functions as both a positive and negative regulator of Notch signaling depending on cellular context . This dual function may complicate interpretation of results and necessitates careful experimental design with appropriate controls.

How can researchers optimize DTX2 antibody performance in Western blot applications?

Optimizing Western blot protocols for DTX2 detection requires attention to several key factors:

• Sample preparation: For complete extraction of DTX2 from both nuclear and cytoplasmic compartments , use lysis buffers containing 1% NP-40 or Triton X-100 with protease inhibitors.

• Protein loading: Load 20-50μg of total protein per lane, with higher amounts potentially needed for tissues with low DTX2 expression.

• Blocking conditions: 5% non-fat dry milk in TBST is typically effective, though 5% BSA may provide lower background for phosphorylation-specific detection.

• Antibody dilution: Start with the manufacturer's recommended dilution (1:100-1:500) and optimize based on signal-to-noise ratio.

• Incubation conditions: Primary antibody incubation at 4°C overnight typically yields better results than shorter incubations at room temperature.

• Detection system: Enhanced chemiluminescence (ECL) detection systems with appropriate sensitivity for the expected expression level.

• Positive control: Include A-549 cell lysate as a positive control, as it has been verified for DTX2 expression .

• Molecular weight verification: Confirm band specificity at the expected 67kDa size .

What approaches are recommended for studying DTX2's role in Notch signaling regulation?

To elucidate DTX2's complex role in Notch signaling regulation, researchers should consider these methodological approaches:

• Genetic manipulation: Utilize CRISPR/Cas9 to create DTX2 knockout or knockin cell lines to study gain or loss of function effects on Notch signaling.

• Reporter assays: Implement Notch-responsive luciferase reporters to quantitatively assess how DTX2 modulation affects downstream transcriptional activity.

• Co-immunoprecipitation studies: Use DTX2 antibodies to identify interacting partners within the Notch pathway, including potential substrates for ubiquitination.

• Domain mapping: Create DTX2 constructs with specific domain deletions or mutations to determine which regions are responsible for its dual regulatory roles in Notch signaling.

• Context-dependent analysis: Compare DTX2 function across different cell types and developmental stages, as DTX2 can function as both a positive and negative regulator depending on context .

• Transcriptional profiling: Analyze changes in Notch target gene expression following DTX2 modulation using RNA-seq or qPCR arrays.

• Protein stability assays: Investigate whether DTX2 affects the stability of Notch pathway components through cycloheximide chase experiments.

This multi-faceted approach addresses DTX2's complex role in mediating the antineural activity of Notch and its context-dependent function as both a positive and negative regulator .

What strategies can researchers employ to detect post-translational modifications of DTX2?

Investigating post-translational modifications (PTMs) of DTX2 requires specialized techniques:

• Phosphorylation analysis:

  • Use phospho-specific antibodies if available

  • Perform immunoprecipitation with DTX2 antibody followed by Western blot with anti-phospho-Ser/Thr/Tyr antibodies

  • Treat samples with phosphatase inhibitors during extraction

  • Consider phospho-enrichment methods prior to mass spectrometry

• Ubiquitination detection:

  • Express tagged ubiquitin (HA or FLAG) and immunoprecipitate under denaturing conditions

  • Use DTX2 antibodies for immunoprecipitation followed by ubiquitin antibody detection

  • Treat cells with proteasome inhibitors (MG132) to accumulate ubiquitinated species

• SUMOylation and other modifications:

  • Similar approaches to ubiquitination, using specific antibodies against SUMO, acetyl groups, etc.

  • Mass spectrometry following enrichment for the specific modification

• Site-directed mutagenesis validation:

  • Once PTM sites are identified, create point mutations at these sites to confirm functional significance

• Kinase/enzyme prediction and validation:

  • Use bioinformatic tools to predict enzymes responsible for DTX2 modifications

  • Validate through kinase inhibitors or enzyme knockdown experiments

These approaches can help elucidate how PTMs regulate DTX2's dual function in Notch signaling and its E3 ligase activity in different cellular contexts.

How can DTX2 antibodies be employed in immunohistochemistry for tissue-specific expression analysis?

Optimizing immunohistochemistry protocols for DTX2 detection in different tissues requires careful methodological consideration:

• Fixation optimization:

  • For formalin-fixed paraffin-embedded (FFPE) tissues, test different fixation times (12-24 hours)

  • Consider alternative fixatives for challenging tissues

  • For nuclear detection, ensure adequate permeabilization given DTX2's dual nuclear-cytoplasmic localization

• Antigen retrieval:

  • Compare heat-induced epitope retrieval methods (citrate buffer pH 6.0 vs. EDTA buffer pH 9.0)

  • Optimize retrieval time (10-30 minutes) based on tissue type

  • Consider enzymatic retrieval for fibrous tissues

• Antibody conditions:

  • Start with manufacturer's recommended dilution range (1:50-1:200 for IHC-P)

  • Test extended incubation times (overnight at 4°C) for weak signals

  • Consider signal amplification systems for low abundance detection

• Controls:

  • Include positive control tissues (based on known DTX2 expression patterns)

  • Include negative controls (primary antibody omission, isotype controls)

  • Consider using tissues from DTX2 knockout models if available

• Counterstaining and analysis:

  • Select counterstains that don't obscure DTX2 detection

  • Consider dual immunofluorescence to co-localize DTX2 with Notch pathway components

  • Implement digital image analysis for quantification of expression levels

These methodological optimizations are essential for accurate assessment of DTX2 expression patterns across different tissues and disease states.

What are the key technical parameters researchers should consider when selecting a DTX2 antibody?

When selecting a DTX2 antibody for research applications, consider these critical technical specifications:

These specifications provide crucial information for experimental design and optimization, ensuring reliable detection of DTX2 in research settings.

How should researchers address cross-reactivity concerns with DTX2 antibodies?

Addressing potential cross-reactivity is critical for generating reliable results with DTX2 antibodies:

• Sequence homology assessment: Analyze the immunogen sequence for similarity to other proteins, particularly other Deltex family members (DTX1, DTX3, DTX4) which share structural domains.

• Multi-validation approach: Implement a comprehensive validation strategy as recommended in modern antibody research :

  • Test antibody in knockout/knockdown systems

  • Compare results with orthogonal detection methods

  • Validate using complementary antibodies targeting different DTX2 epitopes

• Computational prediction: Newer biophysics-informed models can help predict antibody specificity profiles and identify potential cross-reactivity issues . These computational approaches can:

  • Disentangle multiple binding modes associated with specific ligands

  • Design antibodies with customized specificity profiles

  • Mitigate experimental artifacts and biases in selection experiments

• Peptide competition assays: Pre-incubate the antibody with excess immunizing peptide to confirm signal specificity.

• Western blot analysis: Verify a single band at the expected molecular weight (67kDa for DTX2) without additional bands that might indicate cross-reactivity.

• Specificity in multiplex studies: When performing co-localization studies with other Deltex family members, carefully validate antibody specificity to prevent misinterpretation of results.

Implementing these approaches helps ensure that observed signals genuinely represent DTX2 rather than related proteins or non-specific binding.

What are common challenges when working with DTX2 antibodies and how can they be addressed?

Researchers frequently encounter several challenges when working with DTX2 antibodies. Here are methodological solutions for each:

• Weak or absent signal in Western blots:

  • Increase protein loading (up to 50-75μg)

  • Optimize antibody concentration beyond recommended ranges

  • Extend primary antibody incubation to overnight at 4°C

  • Use more sensitive detection systems (enhanced ECL)

  • Ensure sample preparation maintains protein integrity with appropriate protease inhibitors

• High background in immunostaining:

  • Increase blocking time and concentration (5-10% serum/BSA)

  • Use more stringent washing conditions (higher salt concentration in TBST)

  • Titrate primary antibody to lower concentrations

  • Pre-absorb antibody with tissue lysates from irrelevant species

  • Consider using specialized blocking reagents for endogenous biotin or peroxidase

• Nuclear vs. cytoplasmic detection inconsistencies:

  • Optimize fixation and permeabilization protocols specifically for nuclear proteins

  • Consider dual immunofluorescence with known nuclear and cytoplasmic markers

  • Compare different fixatives (formaldehyde vs. methanol) which can affect epitope accessibility

• Variability between experimental replicates:

  • Standardize lysate preparation protocols

  • Prepare large batches of antibody dilutions to use across experiments

  • Include consistent positive controls (A-549 cells)

  • Implement quantitative analysis methods with appropriate normalization

• Discrepancies between antibody lots:

  • Validate each new lot against previous lots

  • Maintain frozen aliquots of validated antibody lots for critical experiments

  • Document lot-specific optimal conditions

These troubleshooting approaches can significantly improve experimental outcomes when working with DTX2 antibodies across different applications.

How can researchers design controlled experiments to validate DTX2 antibody specificity?

Designing rigorous controlled experiments to validate DTX2 antibody specificity is essential for reliable research:

• Genetic validation controls:

  • CRISPR/Cas9 knockout of DTX2 to create true negative controls

  • siRNA knockdown with titrated reduction of DTX2 expression

  • Overexpression systems with tagged DTX2 for positive controls

• Sequential experimental validation:

  • Initial validation in well-characterized cell lines (e.g., A-549)

  • Secondary validation in relevant experimental models

  • Final validation in target experimental system

• Orthogonal detection approaches:

  • Compare antibody-based detection with mRNA expression data

  • Verify localization patterns using fluorescent protein fusions

  • Confirm protein interactions using multiple detection methods

• Cross-antibody validation:

  • Test multiple antibodies targeting different DTX2 epitopes

  • Compare monoclonal and polyclonal antibodies for consistent results

• Biophysics-informed modeling:

  • Implement computational models that can predict antibody specificity

  • Use phage display experiments to validate binding specificity

  • Apply machine learning approaches to optimize antibody selection

These controlled experimental designs align with modern antibody validation frameworks and help ensure that experimental observations genuinely reflect DTX2 biology rather than technical artifacts.

What emerging technologies are enhancing DTX2 antibody research?

Several cutting-edge technologies are advancing the capabilities and applications of DTX2 antibody research:

• Biophysics-informed antibody modeling:

  • Computational models now enable prediction of antibody specificity profiles beyond those observed experimentally

  • These models can identify and disentangle multiple binding modes associated with specific ligands

  • Researchers can generate antibody variants with customized specificity profiles

• Single-cell applications:

  • Optimized protocols for single-cell Western blotting to detect DTX2 in rare cell populations

  • Integration with single-cell RNA-seq for correlation between protein and transcript levels

  • Development of highly sensitive proximity ligation assays for detecting DTX2 interactions in situ

• Live-cell imaging techniques:

  • Nanobody-based approaches for real-time visualization of DTX2 trafficking

  • FRET-based biosensors to monitor DTX2 interactions with Notch pathway components

  • Optogenetic tools to manipulate DTX2 function with spatiotemporal precision

• High-throughput screening platforms:

  • Automated validation of DTX2 antibodies across tissue microarrays

  • Development of DTX2-targeted proteolysis targeting chimeras (PROTACs)

  • CRISPR screens to identify context-dependent roles of DTX2 in different cell types

These emerging technologies will expand our understanding of DTX2's functions in Notch signaling regulation and potentially reveal new therapeutic opportunities in diseases where this pathway is dysregulated.