DSTYK Antibody

What types of DSTYK antibodies are available for research applications?

Several validated DSTYK antibodies are available for research use, with variations in host species, clonality, and target epitopes. The most commonly utilized include rabbit polyclonal antibodies targeting different regions of the DSTYK protein . For instance, antibody A38202 is a rabbit polyclonal that detects endogenous levels of total DSTYK protein, developed using a fusion protein corresponding to residues near the C-terminal region of human dual serine/threonine and tyrosine protein kinase . Other options include antibodies A48282 and A38291, which offer similar reactivity profiles but may have different optimal applications . Researchers should select antibodies based on their specific experimental requirements, target species, and intended applications.

How do I determine the specificity of a DSTYK antibody for my research?

Validating antibody specificity is critical for obtaining reliable experimental results. For DSTYK antibodies, several complementary approaches are recommended:

• Western blot analysis using positive control samples such as 823 and HepG2 cells, which are known to express DSTYK endogenously

• Comparison of staining patterns in wild-type versus DSTYK knockout or knockdown systems

• Peptide competition assays to confirm epitope specificity

• Immunofluorescence microscopy to assess subcellular localization patterns, which should show predominantly cytoplasmic distribution

• Cross-validation using antibodies targeting different epitopes of DSTYK

In studies of genetic disorders involving DSTYK mutations, comparing antibody labeling between affected and unaffected tissues can provide additional validation, as demonstrated in SPG23 patient samples which showed markedly reduced DSTYK labeling .

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

For successful Western blot detection of DSTYK, the following methodological parameters are recommended:

• Sample preparation: 40 μg of total protein lysate per lane provides sufficient detection sensitivity

• Gel percentage: 6% SDS-PAGE is optimal for resolving DSTYK protein

• Primary antibody dilution: 1:200 dilution is typically effective for rabbit polyclonal anti-DSTYK antibodies

• Secondary antibody: Anti-rabbit HRP-conjugated secondary antibody at 1:8000 dilution

• Exposure time: Approximately 1 minute with standard ECL detection systems

• Positive controls: 823 and HepG2 cell lysates serve as reliable positive controls

Reducing non-specific binding may require optimization of blocking conditions and washing steps. For challenging applications, overnight incubation with primary antibody at 4°C may improve signal quality.

How should DSTYK antibodies be applied in immunohistochemistry protocols?

For optimal immunohistochemical detection of DSTYK in formalin-fixed, paraffin-embedded tissues:

• Antigen retrieval: Heat-induced epitope retrieval in citrate buffer (pH 6.0) is generally effective

• Antibody dilution: For rabbit polyclonal antibodies, dilutions of 1:25 to 1:100 are recommended

• Incubation conditions: Overnight incubation at 4°C typically yields best results

• Detection system: Polymer-based detection systems provide good signal-to-noise ratio

• Positive control tissues: Human liver cancer tissue serves as an appropriate positive control

• Counterstaining: Hematoxylin counterstaining allows visualization of tissue architecture

When interpreting IHC results, cytoplasmic staining is the expected pattern for DSTYK . Validation using multiple antibodies targeting different epitopes may be necessary for conclusive results, particularly in tissues with suspected alterations in DSTYK expression.

What considerations are important when using DSTYK antibodies for immunofluorescence applications?

For immunofluorescence detection of DSTYK:

• Fixation method: 4% paraformaldehyde provides good antigen preservation

• Permeabilization: 0.1-0.2% Triton X-100 allows antibody access to intracellular epitopes

• Blocking: 5-10% normal serum from the same species as the secondary antibody reduces background

• Primary antibody dilution: Start with manufacturer recommendations (typically 1:50-1:200)

• Controls: Include both N-terminal and C-terminal targeting antibodies for validation

• Co-staining: Consider co-staining with organelle markers (mitochondria, lysosomes) to assess subcellular localization

Research on SPG23 patients has demonstrated the value of using both N-terminal and C-terminal antibodies in parallel to confirm specificity, particularly when studying potential truncating mutations or deletions in DSTYK .

How does DSTYK function in cancer biology, particularly in lung cancer?

DSTYK has been identified as a novel actionable target in non-small cell lung cancer with significant implications for tumor biology and treatment response . Key findings include:

These findings position DSTYK as both a prognostic biomarker and a potential therapeutic target for improving immunotherapy responses in lung cancer.

What are the neurological implications of DSTYK function based on knockout models?

Studies using DSTYK knockout mice have revealed important neurological functions:

• DSTYK is highly expressed in most brain areas, suggesting significant neurological roles

• A DSTYK knockout mouse line with ablation of the C-terminal region (including the kinase domain) was generated to study its physiological function

• Phenotypic analysis revealed:

  • Knockout mice are viable and fertile

  • No significant gross morphological defects were detected by Nissl staining

  • Basic motor function and balance capacity remained intact as assessed by open field and rotarod tests

• Notably, DSTYK homozygous knockout mice demonstrated impaired learning and memory capabilities compared to heterozygous mice in water maze tests

These findings suggest that while DSTYK is not essential for neurological development, it plays important roles in cognitive functions related to learning and memory, providing valuable insights for researchers studying neurological disorders.

What is known about DSTYK's role in genetic skin disorders and cellular stress responses?

Research has identified DSTYK mutations in autosomal recessive genetic disorders affecting the skin:

• A large intragenic deletion in DSTYK has been established as the molecular basis for three families with SPG23, an autosomal-recessive disorder

• Immunofluorescence microscopy using both N-terminal and C-terminal antibodies showed markedly reduced DSTYK labeling in affected individuals' skin samples

• Ultrastructural analysis revealed:

  • Focal loss of melanocytes

  • Swollen mitochondria and cytoplasmic vacuoles in remaining melanocytes

  • Enlarged mitochondria with abnormal cristae in other cell types including fibroblasts and keratinocytes

• Functional studies demonstrated:

  • Loss of DSTYK in fibroblasts, keratinocytes, and melanocytes leads to increased susceptibility to apoptosis

  • DSTYK plays a predominant role in suppressing caspase-dependent apoptosis in response to UV stress

  • This function occurs across multiple dermal cell types

These findings establish DSTYK as an important regulator of cellular stress responses and apoptosis protection in skin cells, with implications for understanding both genetic disorders and normal skin physiology.

How can DSTYK be targeted for therapeutic development, particularly in cancer immunotherapy?

Recent research has positioned DSTYK as a promising therapeutic target with particular relevance to cancer immunotherapy:

• In lung cancer models, DSTYK inhibition:

  • Sensitizes cancer cells to TNF-α–mediated CD8+ T cell killing

  • Enhances responsiveness of immune-resistant tumors to anti-PD-1 treatment

• Therapeutic development strategies may include:

  • Small molecule inhibitors targeting the kinase domain

  • Biologics that disrupt protein-protein interactions

  • Genetic approaches such as siRNA or CRISPR-based knockdown

• Biomarker development:

  • DSTYK copy number status could serve as a predictive biomarker for immunotherapy response

  • Expression levels might guide patient selection for combination therapies

• Key considerations for therapeutic development include:

  • Selectivity against related kinases

  • Cell permeability for targeting intracellular DSTYK

  • Pharmacokinetic and safety profiles

  • Potential combination strategies with existing immunotherapies

The positioning of DSTYK at the intersection of autophagy, mitochondrial function, and immune response makes it particularly intriguing for development of combination approaches that might expand the percentage of cancer patients benefiting from immune-based treatments.

What methodological approaches are most effective for studying DSTYK-dependent autophagy and mitochondrial functions?

Given DSTYK's established roles in autophagy regulation and mitochondrial function, specialized methodological approaches are recommended:

• For autophagy studies:

  • LC3-II/LC3-I conversion analysis by Western blot with and without lysosomal inhibitors

  • Fluorescence microscopy of GFP-LC3 puncta formation

  • Transmission electron microscopy to visualize autophagosomes and autolysosomes

  • Assessment of autophagy flux using tandem mRFP-GFP-LC3 constructs

  • Quantification of p62/SQSTM1 levels as markers of autophagy completion

• For mitochondrial function analysis:

  • Transmission electron microscopy to assess mitochondrial morphology and cristae structure

  • Live-cell imaging with mitochondrial-targeted fluorescent probes

  • Oxygen consumption rate measurement using Seahorse XF analyzers

  • Mitochondrial membrane potential assessment using JC-1 or TMRM dyes

  • Analysis of mitophagy markers including PINK1 and Parkin recruitment

• For studying DSTYK-specific effects:

  • CRISPR/Cas9-mediated gene editing to generate DSTYK knockouts

  • shRNA or siRNA-mediated knockdown approaches for temporary depletion

  • Rescue experiments with wild-type or mutant DSTYK constructs

  • Pharmacological inhibition when specific inhibitors become available

These methodological approaches, when combined with appropriate DSTYK antibody-based detection methods, provide a comprehensive toolkit for investigating DSTYK's roles in cellular homeostasis and stress responses.

How can contradictory findings about DSTYK's role in cell death pathways be reconciled?

The literature contains apparently contradictory findings regarding DSTYK's role in cell death regulation:

• Some studies suggest DSTYK functions as a positive regulator of both caspase-dependent and -independent cell death pathways when overexpressed in HEK293 cells

• Conversely, recent research in skin cells demonstrates that DSTYK plays a predominant role in suppressing caspase-dependent apoptosis in response to UV stress

These discrepancies can potentially be reconciled through consideration of:

• Cellular context:

  • Different cell types may utilize DSTYK in opposing ways within death pathways

  • Tissue-specific binding partners might alter DSTYK function

• Experimental conditions:

  • Overexpression systems versus endogenous protein studies

  • Presence or absence of external stress stimuli (e.g., UV exposure)

  • Acute versus chronic modulation of DSTYK levels

• Methodological approaches:

  • Different detection methods for quantifying apoptosis

  • Varying time points of analysis after stimulation

• Resolution strategies:

  • Direct comparison studies in multiple cell types

  • Careful analysis of dose-dependent effects

  • Consideration of compensatory mechanisms in different genetic backgrounds

Future research should systematically evaluate DSTYK's role across multiple cell types under standardized conditions, with careful attention to kinetics, dose-dependence, and specific stimuli involved in triggering cell death pathways.

What strategies can address non-specific binding when using DSTYK antibodies?

Non-specific binding is a common challenge with antibody-based detection methods. For DSTYK antibodies, consider these application-specific troubleshooting approaches:

• For Western blot applications:

  • Optimize blocking conditions (5% non-fat dry milk or BSA in TBST)

  • Increase washing duration and frequency

  • Use antigen-affinity purified antibodies when available

  • Titrate primary antibody concentration (starting with 1:200 dilution)

  • Confirm gel percentage is appropriate (6% SDS-PAGE recommended)

  • Include positive controls (823 and HepG2 cell lysates)

• For immunohistochemistry:

  • Optimize antigen retrieval methods

  • Use dilutions of 1:25 to 1:100 as starting points

  • Include appropriate blocking of endogenous peroxidases and biotin

  • Utilize polymer-based detection systems

  • Include both positive and negative control tissues

  • Compare N-terminal and C-terminal antibodies to confirm specificity

• General considerations:

  • Validate antibody specificity using DSTYK knockout or knockdown systems

  • Consider peptide competition assays

  • Use multiple antibodies targeting different epitopes

These strategies should help minimize non-specific binding and improve signal-to-noise ratio in DSTYK detection applications.

How should researchers interpret divergent results from different DSTYK antibodies?

When different DSTYK antibodies yield contradictory results, a systematic approach to interpretation is essential:

• Consider epitope location:

  • N-terminal versus C-terminal epitopes may yield different results in samples with truncating mutations

  • Isoform-specific epitopes might detect distinct subsets of DSTYK proteins

• Validation strategies:

  • Compare antibody performance in known positive and negative control samples

  • Use genetic models (knockouts, knockdowns) to confirm specificity

  • Employ complementary techniques (RNA analysis, mass spectrometry)

• Experimental context:

  • Different applications (WB, IHC, IF) may require different antibodies

  • Fixation and sample preparation can affect epitope accessibility

  • Post-translational modifications might mask certain epitopes

• Resolution approach:

  • Create a validation matrix comparing multiple antibodies across standardized samples

  • Document specific conditions where discrepancies occur

  • Consider the possibility of tissue-specific isoforms or modifications

The case of SPG23 patients illustrates this principle, where both N-terminal and C-terminal antibodies were used to confirm reduced DSTYK expression, providing stronger evidence than either antibody alone would have provided .