DTX13 Antibody

What is DTX1 and why is it important in research?

DTX1 (deltex E3 ubiquitin ligase 1) is a crucial protein involved in ubiquitination processes and Notch signaling regulation. It is also known as deltex 1, hDx-1, RNF140, E3 ubiquitin-protein ligase DTX1, and RING-type E3 ubiquitin transferase DTX1. The protein has a molecular weight of approximately 67.4 kilodaltons . DTX1 is significant in research because it functions as a cytoplasmic and nuclear ubiquitin ligase that interacts with the Notch-1 intracellular domain and regulates Notch-induced gene transcription . The protein contains two WWE domains (amino acids 14-94 and 95-171) and one RING-type zinc finger (amino acids 411-472), which are critical for its biological functions . DTX1 represents an important research target due to its involvement in multiple signaling pathways and disease associations.

What species orthologs exist for DTX1 and how conserved is the protein?

DTX1 is highly conserved across multiple species, making it amenable to comparative studies. Based on gene homology, researchers have identified orthologs in various mammalian species including canine, porcine, monkey, mouse, and rat models, as well as some plant species . Within amino acids 1-147, human Deltex-1 shares remarkable 97% amino acid sequence identity with mouse and rat Deltex-1 . This high degree of conservation suggests evolutionary importance and enables researchers to effectively use animal models for studying DTX1 functions. When selecting antibodies for cross-species research, it's advisable to verify the specific reactivity profile of your chosen antibody, as commercial options vary in their species recognition capabilities.

How should researchers select the appropriate DTX1 antibody for their specific experiments?

When selecting DTX1 antibodies, researchers should consider several factors:

• Experimental application: Different antibodies are optimized for specific techniques such as Western Blot (WB), Immunohistochemistry (IHC), Immunocytochemistry (ICC), ELISA, or Flow Cytometry .

• Species reactivity: Verify compatibility with your experimental model. Some antibodies react with human DTX1 only, while others recognize mouse, rat, rabbit, bovine, and other species .

• Epitope recognition: Consider whether the antibody targets the N-terminal region, C-terminal region, or specific domains within DTX1 .

• Validation data: Review published validation data demonstrating the antibody's specificity and performance in applications similar to your planned experiments .

• Format: Determine if you need unconjugated antibodies or those conjugated with specific tags for your detection method .

The commercial landscape offers 229 different DTX1 antibody products across 19 suppliers, providing options for virtually any research application .

What are the optimal protocols for detecting DTX1 using Western blot analysis?

For optimal Western blot detection of DTX1:

• Sample preparation: Lysates from K562 human chronic myelogenous leukemia cell line or SW13 human adrenal cortex adenocarcinoma cell line have been successfully used to detect DTX1 .

• Membrane and conditions: Use PVDF membrane under reducing conditions with appropriate immunoblot buffer (e.g., Immunoblot Buffer Group 1) .

• Antibody concentration: Probe with 2 μg/mL of anti-DTX1 antibody, followed by appropriate HRP-conjugated secondary antibody .

• Detection: Look for a specific band at approximately 67 kDa, which corresponds to the DTX1 protein .

• Controls: Include positive controls (K562 or SW13 cell lines) and negative controls to ensure specificity .

The Western blot technique allows for semi-quantitative analysis of DTX1 expression levels across different experimental conditions or tissue types.

How can researchers effectively use immunocytochemistry to localize DTX1 in cells?

For effective immunocytochemistry to visualize DTX1 cellular localization:

• Cell preparation: Use cell types known to express DTX1, such as K562 human chronic myelogenous leukemia cells .

• Fixation method: Perform immersion fixation to maintain cellular architecture .

• Antibody concentration: Apply anti-DTX1 antibody at 10 μg/mL for 3 hours at room temperature .

• Visualization: Use fluorescently conjugated secondary antibodies (e.g., NorthernLights™ 557-conjugated Anti-Mouse IgG) .

• Counterstaining: Counter-stain nuclei with DAPI for cellular context .

• Analysis: Observe for specific staining in the cytoplasm, which is the primary localization site for DTX1 .

This approach enables researchers to determine subcellular localization patterns of DTX1, which may change in different cellular contexts or disease states.

What considerations are important when using flow cytometry to detect DTX1?

When using flow cytometry for DTX1 detection:

• Cell preparation: Use suitable cell lines such as K562 human chronic myelogenous leukemia cells .

• Fixation and permeabilization: Fix cells with paraformaldehyde and permeabilize with saponin to allow antibody access to intracellular DTX1 .

• Antibody selection: Choose a validated anti-DTX1 antibody with demonstrated flow cytometry applications .

• Controls: Include isotype control antibodies to establish background staining levels .

• Secondary detection: Use appropriate secondary antibodies such as Allophycocyanin-conjugated Anti-Mouse IgG .

• Analysis parameters: Set appropriate gating strategies to distinguish positive from negative populations.

Flow cytometry offers quantitative assessment of DTX1 expression across cell populations and enables correlation with other cellular markers.

How is DTX1 implicated in autoimmune disorders and what methods best detect these associations?

DTX1 has emerging significance in autoimmune research, particularly in dermatomyositis:

• Autoantibody associations: Studies of dermatomyositis patients with TIF1γ (TRIM33) autoantibodies have revealed connections to DTX1. Research has identified antibodies against multiple TRIM proteins, potentially related to DTX1 function .

• Detection methodologies: Next-generation sequencing (NGS) approaches have been used to identify autoantibodies against DTX1 and related proteins in patient plasma .

• Disease specificity: The autoantibody profile against DTX1 and other proteins appears to create a disease-specific signature, with dermatomyositis patients showing approximately 2:1 (total proteins) and 3:1 (high specificity) ratios of identified autoantibodies compared to healthy controls .

• Interferon connections: DTX1 appears within networks of interferon-regulated proteins targeted by autoantibodies in dermatomyositis, suggesting its role in interferon signaling pathways .

• Research approach: Immunoglobulin disease-specific epitope-enrichment methods combined with high-throughput identification of microbial and human antigens provide effective tools for studying these associations .

What is the relationship between DTX1 and the Notch signaling pathway?

DTX1 plays a critical role in the Notch signaling pathway:

• Molecular interaction: DTX1 functions as a cytoplasmic and nuclear ubiquitin ligase that directly interacts with the Notch-1 intracellular domain .

• Transcriptional regulation: Through this interaction, DTX1 regulates Notch-induced gene transcription, serving as an important modulator of this pathway .

• Structural basis: The WWE domains and RING-type zinc finger domain in DTX1 are likely involved in protein-protein interactions with Notch pathway components .

• Experimental approaches: Co-immunoprecipitation, reporter gene assays, and protein localization studies can effectively investigate DTX1-Notch interactions.

• Research implications: Understanding this relationship is important for developmental biology research and disease investigations, as Notch signaling dysregulation contributes to multiple pathologies.

How does the ubiquitin ligase activity of DTX1 influence its biological functions?

As an E3 ubiquitin ligase, DTX1's enzymatic activity is central to its biological roles:

• Mechanistic function: DTX1 (E3 ubiquitin-protein ligase DTX1) attaches ubiquitin to target proteins, marking them for degradation or altering their function .

• Substrate identification: Research to identify DTX1 substrates beyond Notch pathway components remains an active area of investigation. Techniques like proximity-dependent biotin identification (BioID) and quantitative proteomics can help identify novel substrates.

• RING domain function: The RING-type zinc finger domain (amino acids 411-472) is critical for its ubiquitin ligase activity .

• Regulatory mechanisms: Studies examining how DTX1's ubiquitin ligase activity is itself regulated provide insights into signaling pathway integration.

• Potential for targeted therapies: Understanding this enzymatic activity may lead to development of specific inhibitors for therapeutic applications.

What are common challenges when working with DTX1 antibodies and how can they be addressed?

Researchers commonly encounter several challenges when working with DTX1 antibodies:

• Cross-reactivity: Due to structural similarities between DTX family members (DTX1, DTX2, DTX3, DTX4), antibodies may cross-react. Solution: Validate specificity using knockout/knockdown controls or cell lines with known DTX expression profiles.

• Low signal intensity: DTX1 may be expressed at low levels in some cell types. Solution: Optimize antibody concentration, increase protein loading for Western blots, or use signal amplification methods.

• Background staining: Non-specific binding can obscure results. Solution: Optimize blocking conditions, titrate antibody concentrations, and include appropriate controls.

• Epitope masking: Protein interactions or post-translational modifications may mask epitopes. Solution: Try different antibodies targeting distinct regions of DTX1 or modify sample preparation methods.

• Storage and handling issues: Antibody degradation affects performance. Solution: Aliquot antibodies to avoid freeze-thaw cycles and follow manufacturer's storage recommendations (typical stability is 12 months at -20 to -70°C, 1 month at 2-8°C after reconstitution) .

How can researchers validate the specificity of DTX1 antibodies?

Validating DTX1 antibody specificity should include:

• Positive and negative controls: Use cell lines with known DTX1 expression (e.g., K562) as positive controls and appropriate negative controls .

• Multiple detection methods: Confirm findings using different techniques (e.g., Western blot, immunostaining, flow cytometry) .

• Competitive blocking: Pre-incubate antibody with purified DTX1 protein to demonstrate binding specificity.

• Genetic validation: Use siRNA knockdown, CRISPR knockout, or overexpression systems to confirm antibody specificity.

• Cross-reactivity testing: Especially important for distinguishing between DTX1 and DTX4, as some antibodies may recognize both proteins .

• Mass spectrometry verification: For definitive identification of immunoprecipitated proteins.

Thorough validation ensures experimental results accurately reflect DTX1 biology rather than artifacts.

What emerging areas of DTX1 research hold promise for translational applications?

Several emerging areas of DTX1 research show translational potential:

• Autoimmune disease biomarkers: The presence of DTX1 autoantibodies in dermatomyositis suggests potential applications as diagnostic or prognostic biomarkers for autoimmune conditions .

• Cancer biology: DTX1's role in Notch signaling, which is dysregulated in many cancers, suggests potential as a therapeutic target. Research in K562 leukemia cells indicates relevance to hematological malignancies .

• Immunomodulation: The connection between DTX1 and interferon-regulated proteins suggests roles in immune response regulation that could be therapeutically exploited .

• Developmental biology: Given its role in Notch signaling, DTX1 research informs understanding of developmental processes and related disorders.

• Drug development: The ubiquitin ligase activity of DTX1 represents a potentially druggable target, with emerging interest in developing specific inhibitors or modulators.

How might single-cell analysis approaches enhance our understanding of DTX1 function?

Single-cell analysis technologies offer new insights into DTX1 biology:

• Expression heterogeneity: Single-cell RNA sequencing can reveal cell-to-cell variability in DTX1 expression within seemingly homogeneous populations.

• Spatial context: Spatial transcriptomics and high-resolution imaging techniques can map DTX1 expression in tissue contexts, revealing microenvironmental influences.

• Temporal dynamics: Live-cell imaging with fluorescently tagged DTX1 can capture dynamic changes in localization and expression.

• Pathway integration: Single-cell multi-omics approaches can correlate DTX1 expression with other molecular features to build comprehensive signaling networks.

• Rare cell populations: Identification of cells with unique DTX1 expression patterns that may be missed in bulk analyses.

These approaches will help resolve contradictory findings from bulk analyses and provide more nuanced understanding of DTX1's contextual functions.