DTX42 Antibody

What is DTX4 and what cellular functions does it perform?

DTX4 belongs to the Deltex family of proteins, which function as E3 ubiquitin ligases that interact with the Notch signaling pathway. Specifically, Deltex-1 (DTX1), a related family member, is an approximately 75 kDa cytoplasmic and nuclear ubiquitin ligase that interacts with the Notch-1 intracellular domain and regulates Notch-induced gene transcription . DTX proteins contain WWE domains and RING-type zinc finger domains that are essential for their function. DTX4 shares structural similarities with DTX1, including these conserved domains that mediate protein-protein interactions and ubiquitination activity. The subcellular localization of DTX proteins typically includes both cytoplasmic and nuclear distribution, as demonstrated by immunofluorescence studies showing specific staining localized to the cytoplasm in various cell lines .

What types of DTX4 antibodies are currently available for research?

Research-grade DTX4 antibodies are available in both monoclonal and polyclonal formats, each with distinct characteristics suited for different experimental applications:

Monoclonal antibodies like MAB7157 recognize specific epitopes and may cross-react with related family members (in this case, both DTX1 and DTX4), while polyclonal antibodies such as 25222-1-AP may recognize multiple epitopes on the target protein, potentially providing stronger signals in certain applications .

What are the optimal conditions for Western blot analysis with DTX4 antibodies?

When performing Western blot analysis with DTX4 antibodies, researchers should consider the following protocol parameters:

For monoclonal antibodies like Human DTX1/DTX4 (MAB7157), a concentration of 2 μg/mL has been validated with PVDF membrane to detect bands at approximately 67 kDa in K562 and SW13 cell lines . These experiments were conducted under reducing conditions using specific immunoblot buffer groups, which is critical for proper protein denaturation and epitope exposure.

For polyclonal antibodies such as DTX4 (25222-1-AP), recommended dilutions range from 1:200 to 1:1000 . Optimization is essential, as the ideal concentration may vary depending on sample type and protein expression levels. When analyzing mouse kidney tissue, this antibody has been successfully used to detect DTX4 .

To ensure optimal results, researchers should implement proper blocking, incubate with the primary antibody at either 4°C overnight or room temperature for 1-3 hours, and use appropriate HRP-conjugated secondary antibodies (such as Anti-Mouse IgG Secondary Antibody for monoclonal antibodies) .

How can I optimize immunofluorescence protocols for DTX4 detection?

For successful immunofluorescence detection of DTX4 in fixed cells and tissues:

When using monoclonal antibodies like MAB7157, a concentration of 10 μg/mL applied for 3 hours at room temperature has been validated for K562 cells . This approach revealed specific cytoplasmic localization of DTX1/DTX4. For visualization, fluorophore-conjugated secondary antibodies such as NorthernLights™ 557-conjugated Anti-Mouse IgG have proven effective, with DAPI counterstaining to mark nuclei .

For polyclonal antibodies such as 25222-1-AP, dilutions ranging from 1:50 to 1:500 for immunofluorescence on paraffin-embedded tissues (IF-P) and 1:10 to 1:100 for immunofluorescence on cultured cells (IF/ICC) are recommended . This antibody has been successfully validated for detecting DTX4 in mouse embryo tissue and HEK-293 cells .

A critical methodological consideration is the fixation and permeabilization protocol. For adherent cells, standard paraformaldehyde fixation followed by detergent permeabilization is typically sufficient. For non-adherent cells like K562, specialized protocols for fluorescent ICC staining may be required to ensure proper preservation of cellular morphology and protein localization .

What methods are available for detecting DTX4 in flow cytometry applications?

Flow cytometry provides a quantitative approach to analyzing DTX4 expression at the single-cell level. Effective protocols include:

For intracellular staining of DTX4, cells (such as K562 human chronic myelogenous leukemia cell line) should be fixed with paraformaldehyde and permeabilized with saponin to allow antibody access to intracellular antigens . Using monoclonal antibodies like DTX1/DTX4 (MAB7157) with appropriate isotype controls (e.g., MAB0041) is essential for determining specific binding. Fluorophore-conjugated secondary antibodies, such as Allophycocyanin-conjugated Anti-Mouse IgG, enable detection of positive populations .

To ensure accurate results, always include:

• Unstained controls

• Isotype controls to assess non-specific binding

• Single-color controls for compensation if using multiple fluorophores

• Titration of antibody concentration to determine optimal signal-to-noise ratio

Sample viability assessment prior to fixation is also critical, as dead cells can contribute to non-specific binding and false-positive signals.

How can I assess and improve DTX4 antibody specificity for closely related Deltex family proteins?

Distinguishing between closely related Deltex family members presents a significant challenge due to their structural similarities. Advanced approaches to address this include:

Antibody validation in knockout/knockdown systems: Utilizing CRISPR/Cas9 or siRNA technology to create DTX4-deficient cell lines provides the most definitive control for antibody specificity testing . This approach has been documented in published literature for DTX4 antibody validation.

Epitope mapping: Understanding the specific regions recognized by DTX4 antibodies is crucial. For instance, monoclonal antibody MAB7157 targets Met1-Phe147 of human DTX1, which may explain its cross-reactivity with DTX4 due to sequence homology in this region . Computational sequence alignment of Deltex family members can help predict potential cross-reactivity.

Biophysics-informed modeling: Recent advancements employ biophysically interpretable models to disentangle multiple binding modes associated with specific ligands . This approach identifies different binding modes associated with particular ligands, enabling the prediction and generation of antibody variants with improved specificity profiles. The model captures the thermodynamics of binding by parameterizing each binding mode through shallow dense neural networks, optimizing parameters globally to reflect antibody population evolution across experiments .

What computational approaches can enhance the design of highly specific DTX4 antibodies?

Computational methodologies have revolutionized antibody design, particularly for achieving discrimination between closely related epitopes:

Recent advances combine high-throughput sequencing with machine learning to predict antibody properties beyond experimentally observed sequences . Biophysics-informed models can now be trained on experimentally selected antibodies to associate distinct binding modes with specific ligands. This approach enables prediction and generation of antibody variants with customized specificity profiles not present in initial libraries .

The computational framework involves:

• Parameterizing binding energies using neural networks for each potential binding mode

• Calculating selection probabilities based on these energies and experimental conditions

• Optimizing parameters to match observed enrichment patterns across multiple selection experiments

• Using the trained model to design new antibodies by optimizing over sequence space

For generating DTX4-specific antibodies, the model would minimize energy functions associated with DTX4 binding while maximizing those associated with other Deltex family members, thereby engineering discriminatory capacity into the antibody design .

How can phage display technology be leveraged to develop highly specific DTX4 antibodies?

Phage display offers a powerful selection platform for developing DTX4-specific antibodies through strategic experimental design:

A more advanced approach utilizes biophysics-informed models trained on phage display data. This involves:

• Creating antibody libraries with systematic variation in complementarity-determining regions (CDRs)

• Performing selections against various ligand combinations

• Sequencing recovered antibodies at each stage

• Training computational models to disentangle binding modes

• Using the model to predict and design antibodies with desired specificity profiles

These models can successfully distinguish between chemically similar ligands even when they cannot be experimentally dissociated from other epitopes present in the selection, making them particularly valuable for developing DTX4-specific antibodies that don't cross-react with other Deltex family members .

What sample preparation techniques optimize DTX4 detection in different experimental contexts?

Optimal sample preparation is critical for reliable DTX4 detection across various experimental platforms:

For Western blotting:
Efficient cell lysis is essential for complete protein extraction. For DTX4 detection, RIPA buffer supplemented with protease inhibitors has proven effective in extracting this approximately 67 kDa protein from cell lines such as K562 and SW13 . Samples should be processed under reducing conditions to properly denature the protein and expose epitopes. Loading 20-50 μg of total protein per lane typically provides sufficient signal while maintaining specificity.

For immunofluorescence:
For adherent cells, fixation with 4% paraformaldehyde for 15-20 minutes followed by permeabilization with 0.1-0.5% Triton X-100 preserves DTX4 localization while allowing antibody access. For tissues, proper antigen retrieval methods (such as citrate buffer heating) may be necessary to unmask epitopes after formalin fixation and paraffin embedding .

For flow cytometry:
Single-cell suspensions should be fixed with paraformaldehyde and permeabilized with saponin specifically for intracellular DTX4 detection . Titration of both primary and secondary antibodies is essential to determine optimal concentrations that maximize specific signal while minimizing background.

How should I troubleshoot inconsistent or unexpected results with DTX4 antibodies?

When encountering problematic results with DTX4 antibodies, systematic troubleshooting should address these common issues:

Non-specific bands in Western blot:

• Increase blocking time/concentration (5% BSA or milk in TBST)

• Optimize antibody dilution (test range from 1:200 to 1:1000)

• Increase washing stringency with higher salt or detergent concentration

• Confirm protein size (DTX4 should appear at approximately 67 kDa)

• Consider using gradient gels for better separation of nearby molecular weight proteins

Weak or absent signal:

• For Western blot, ensure adequate protein loading (30-50 μg total protein)

• Verify transfer efficiency with Ponceau S staining

• Extend primary antibody incubation time (overnight at 4°C)

• Freshly prepare detection reagents

• Confirm DTX4 expression in your cell/tissue type (K562 and SW13 cell lines are positive controls)

High background in immunofluorescence:

• Increase blocking time with specific blocking agents

• Further dilute primary antibody

• Include 0.1% Tween-20 in wash buffers

• Reduce secondary antibody concentration

• Ensure proper fixation and permeabilization

• Include controls omitting primary antibody

What validation approaches should be employed to confirm DTX4 antibody specificity?

Multiple detection methods:
Confirm DTX4 detection using complementary techniques (e.g., Western blot, immunofluorescence, and flow cytometry) to verify consistent protein detection patterns .

Genetic approaches:
CRISPR/Cas9 or siRNA-mediated knockdown/knockout of DTX4 provides the gold standard for antibody validation . Absence or significant reduction of signal in DTX4-depleted samples confirms antibody specificity.

Peptide competition:
Pre-incubation of the antibody with the immunizing peptide should abolish specific binding if the antibody is truly target-specific.

Recombinant protein expression:
Overexpression of tagged DTX4 constructs can serve as positive controls and help distinguish between specific and non-specific signals.

Cross-species reactivity assessment:
The conservation of DTX4 across species can be leveraged for validation. For example, antibodies showing reactivity with human, mouse, and rat DTX4 in patterns consistent with evolutionary conservation provide additional confidence in specificity .

How might emerging technologies enhance DTX4 antibody development and applications?

The landscape of DTX4 antibody research continues to evolve with promising new technological approaches:

Biophysics-informed models represent a significant advancement in antibody engineering, enabling the design of antibodies with customized specificity profiles . These models can predict binding properties beyond the scope of experimentally observed sequences and generate novel antibody sequences with desired binding characteristics. By optimizing energy functions associated with specific binding modes, researchers can develop DTX4 antibodies with either highly specific binding to DTX4 alone or cross-specificity for multiple Deltex family members .

Integration of high-throughput sequencing with phage display experiments provides a powerful methodology for monitoring antibody library composition throughout the selection process . This approach allows for the identification of sequence features associated with specific binding modes, even when dealing with chemically similar ligands that cannot be experimentally dissociated.