DTX2 Antibody

What types of DTX2 antibodies are available for research applications?

Currently, researchers have access to several DTX2 antibodies, including polyclonal antibodies like the Rabbit Polyclonal Antibody (CAB7398) and antibody 18565-1-AP . These antibodies are primarily used in applications such as Western blot (WB), immunohistochemistry (IHC), and ELISA . The DTX2 antibodies available show reactivity primarily with human samples, though some also react with mouse and rat samples . The selection of a specific antibody depends on the intended application and experimental design.

What is the molecular weight and structure of the DTX2 protein that antibodies target?

DTX2 has a calculated molecular weight of approximately 67 kDa, which is consistently observed in Western blot applications . The protein contains sequences corresponding to amino acids 1-280 of human DTX2 (NP_065943.2), which serves as the immunogen for some commercial antibodies . DTX2 harbors three hidden nuclear localization signals and functions as an E3 ubiquitin ligase, which is critical for its role in protein regulation through the ubiquitination pathway .

How do DTX2 antibodies contribute to understanding signaling pathways?

DTX2 antibodies enable researchers to detect and analyze DTX2 expression patterns, which is crucial for understanding its role in the Notch signaling pathway . Through techniques like immunoprecipitation, researchers have demonstrated that DTX2 interacts with helicase-like transcription factor (HLTF), revealing a new regulatory axis in glioma development . Immunofluorescence assays utilizing these antibodies have shown that DTX2 and HLTF co-localize in the nucleus, providing spatial context for their interaction . By facilitating the study of these protein-protein interactions, DTX2 antibodies help elucidate complex signaling networks underlying cancer progression.

What are the optimal conditions for using DTX2 antibodies in Western blot analysis?

For Western blot applications, DTX2 antibodies should be used at dilutions ranging from 1:100 to 1:500 . The optimal protocol involves standard sample preparation, SDS-PAGE separation, transfer to a membrane, blocking, and incubation with the primary antibody overnight at 4°C. When using the DTX2 Rabbit Polyclonal Antibody (CAB7398), researchers should expect to observe a band at approximately 67 kDa, corresponding to the calculated molecular weight of DTX2 . For positive control samples, A-549 cells have been validated . It's important to note that optimization may be required for each specific experimental system to achieve optimal results.

How should DTX2 antibodies be used for immunohistochemistry applications?

For immunohistochemistry applications, DTX2 antibodies should be used at dilutions ranging from 1:20 to 1:200 . Antigen retrieval is a critical step, with recommended protocols including either TE buffer pH 9.0 or citrate buffer pH 6.0 . Positive IHC detection has been validated in human testis tissue and human hepatocirrhosis tissue . The standard protocol involves deparaffinization, rehydration, antigen retrieval, blocking, primary antibody incubation, secondary antibody application, visualization, counterstaining, and mounting. As with all antibody applications, optimization of incubation times and dilutions may be necessary for specific tissue types.

What controls should be included when using DTX2 antibodies in experimental designs?

A comprehensive experimental design using DTX2 antibodies should include several controls:

Inclusion of these controls ensures result reliability and facilitates accurate interpretation of experimental outcomes .

How can researchers effectively use DTX2 antibodies to study protein-protein interactions?

To study DTX2 protein interactions, researchers can employ co-immunoprecipitation (co-IP) assays using DTX2 antibodies . This approach has successfully demonstrated the interaction between DTX2 and HLTF in glioma cells . The protocol involves:

• Preparing whole-cell lysates from cells expressing Flag-tagged DTX2 or empty vector

• Incubating lysates with DTX2 antibody-conjugated beads

• Washing to remove non-specific binding

• Eluting bound proteins

• Analyzing by Western blot for interacting partners

Complementary approaches include immunofluorescence co-localization studies, which have shown that DTX2 and HLTF signals co-localize in the nucleus . For advanced studies, proximity ligation assays can provide higher resolution detection of protein interactions in situ.

How can researchers investigate DTX2-mediated ubiquitination using DTX2 antibodies?

Investigating DTX2-mediated ubiquitination requires specialized experimental approaches leveraging DTX2 antibodies. Researchers should implement in vitro ubiquitination assays by:

• Immunoprecipitating DTX2 from cells using DTX2 antibodies

• Incubating the purified DTX2 with potential substrate proteins (e.g., HLTF)

• Adding ubiquitin, E1, and E2 enzymes in an ATP-containing buffer

• Analyzing ubiquitination by Western blot using anti-ubiquitin antibodies

This approach has successfully demonstrated that DTX2 can downregulate HLTF protein levels by increasing ubiquitination of HLTF protein . For in vivo ubiquitination studies, researchers can treat cells with proteasome inhibitors before immunoprecipitation to prevent degradation of ubiquitinated proteins. Subsequent Western blot analysis with both DTX2 and target protein antibodies can reveal ubiquitination patterns.

What experimental designs can reveal the role of DTX2 in cancer progression?

Comprehensive investigation of DTX2's role in cancer progression requires multi-level experimental approaches:

These approaches collectively provide insight into DTX2's oncogenic functions and potential as a therapeutic target .

How can researchers assess the relationship between DTX2 expression and clinical outcomes?

To assess correlations between DTX2 expression and clinical outcomes, researchers should implement bioinformatic analysis of publicly available datasets (such as The Cancer Genome Atlas) combined with direct tissue analysis . Key methodological approaches include:

• Immunohistochemical analysis of DTX2 expression in patient tumor samples using validated antibodies at optimized dilutions (1:50-1:200)

• Correlation of expression levels with patient survival data using Kaplan-Meier analysis

• Multivariate analysis to control for confounding factors

• Assessment of co-expression with interacting partners like HLTF

What considerations should researchers take when studying DTX2 across different cancer types?

When investigating DTX2 across cancer types, researchers should consider several factors:

• Tissue-specific expression patterns and functions of DTX2

• Variability in antibody performance across different tissue types

• Potential differences in DTX2 post-translational modifications

• Cancer-specific binding partners and regulatory networks

• Differential subcellular localization of DTX2 (cytoplasm vs. nucleus)

Researchers should validate antibody specificity for each cancer type using positive controls such as DTX2-overexpressing cells and negative controls like DTX2-knockdown cells . Comparative studies should maintain consistent experimental conditions, including antibody dilutions, incubation times, and detection methods, to ensure valid cross-cancer comparisons.

What are common issues with DTX2 antibody detection and how can they be resolved?

Common challenges and solutions when working with DTX2 antibodies include:

Researchers should systematically optimize each parameter while maintaining appropriate controls to ensure reliable and reproducible results.

How should researchers interpret conflicting results from different DTX2 antibodies?

When faced with conflicting results from different DTX2 antibodies, researchers should:

• Compare antibody specifications, including immunogens and epitopes recognized

• Validate each antibody using positive and negative controls (DTX2-overexpressing and knockdown cells)

• Perform cross-validation with orthogonal techniques (e.g., mass spectrometry)

• Consider the possibility of detecting different DTX2 isoforms or post-translationally modified forms

• Evaluate the specificity of each antibody through immunoprecipitation followed by mass spectrometry

Different antibodies may recognize distinct epitopes that could be differentially accessible depending on protein conformation, complex formation, or post-translational modifications. When publishing results, researchers should clearly specify which antibody was used and under what conditions to facilitate result interpretation and reproducibility.

What storage and handling practices maximize DTX2 antibody performance and longevity?

To maintain optimal DTX2 antibody performance:

• Store antibodies at -20°C in appropriate buffer conditions (e.g., PBS with 0.02% sodium azide and 50% glycerol pH 7.3)

• Avoid repeated freeze-thaw cycles by preparing small aliquots

• Store working dilutions at 4°C for short-term use only (1-2 weeks)

• Check expiration dates and stability information provided by manufacturers

• Follow specific storage recommendations for each antibody product

Some DTX2 antibodies remain stable for one year after shipment when properly stored . For the 20μl size of certain products, it's worth noting that they may contain 0.1% BSA which can enhance stability . Proper storage and handling significantly impact experimental reproducibility and reliability.

How should researchers quantify and normalize DTX2 expression levels in experimental studies?

For accurate quantification of DTX2 expression:

• For Western blot analysis:

  • Use appropriate loading controls (β-actin, GAPDH, or total protein stains)

  • Employ densitometric analysis software for band intensity quantification

  • Normalize DTX2 signal intensity to loading control

  • Include a standard curve using recombinant DTX2 for absolute quantification

• For immunohistochemistry:

  • Use validated scoring systems (H-score, Allred score, or percentage of positive cells)

  • Employ digital image analysis software for unbiased quantification

  • Include control tissues on the same slide for normalization

  • Use multiple independent observers to reduce subjective bias

• For qPCR analysis of DTX2 mRNA:

  • Normalize to multiple reference genes

  • Use the 2^(-ΔΔCT) method for relative quantification

Data should be presented with appropriate statistical analysis, including measures of central tendency and dispersion, with significance testing between experimental groups.

What insights can DTX2/HLTF axis analysis provide in cancer research?

Analysis of the DTX2/HLTF axis offers several valuable insights:

• Mechanistic understanding of oncogenic pathways: DTX2 negatively regulates HLTF through ubiquitination, identifying a novel regulatory mechanism in cancer progression .

• Prognostic value: The negative correlation between DTX2 and HLTF expression (r = -0.6031, p < 0.01) in glioma tissues suggests potential use as a prognostic indicator .

• Therapeutic targeting opportunities: Understanding the DTX2/HLTF interaction may reveal new strategies for intervention in glioma and potentially other cancers .

• Biomarker development: The DTX2/HLTF axis could serve as a prognostic or therapeutic marker for patient stratification .

• Cell biology insights: This axis reveals how E3 ubiquitin ligases like DTX2 regulate protein stability and function in cancer contexts .

Research has demonstrated that DTX2 knockdown suppresses glioma cell proliferation and migration through HLTF, as evidenced by rescue experiments where double knockdown of DTX2 and HLTF restored the tumorigenic phenotype .

How can researchers effectively visualize and present DTX2 antibody-based experimental data?

For effective presentation of DTX2 antibody-based experimental data:

• Western blot data:

  • Show full blots with molecular weight markers

  • Include all relevant controls

  • Present quantification with statistical analysis

  • Use consistent scaling and cropping across experimental groups

• Immunohistochemistry/Immunofluorescence:

  • Include representative images at appropriate magnifications

  • Show both overview and high-magnification images

  • Present co-localization data with appropriate overlap metrics

  • Include scale bars and indicate magnification

• Functional assays:

  • Present data in standardized formats (bar graphs, box plots)

  • Include individual data points when possible

  • Clearly indicate statistical tests used and significance levels

  • Use consistent color schemes for experimental groups across figures

When presenting DTX2/HLTF interaction data, researchers should consider showing both proteins in the same figure panel to facilitate comparison, as demonstrated in studies showing the negative correlation between these proteins in glioma tissues .

How can researchers integrate DTX2 antibody-based findings with other molecular data?

To generate comprehensive insights, researchers should integrate DTX2 antibody-based findings with:

• Transcriptomic data: Correlate DTX2 protein expression with mRNA levels from RNA-seq or microarray data

• Genomic data: Investigate associations between DTX2 protein expression and genetic alterations

• Clinical data: Integrate DTX2 expression patterns with patient outcomes, treatment responses, and clinical parameters

• Pathway analysis: Place DTX2 findings within broader signaling networks, particularly Notch pathway components

• Interactome data: Map DTX2 protein interactions using data from immunoprecipitation studies combined with mass spectrometry

This multi-omics approach provides a systems-level understanding of DTX2 function. For instance, combining DTX2 protein expression data with transcriptomic profiling can reveal whether regulation occurs at transcriptional or post-transcriptional levels, while integration with clinical data can identify patient subgroups where DTX2 may have particular diagnostic or therapeutic relevance.