DRAXIN Antibody

What is DRAXIN and why is it significant in neurodevelopmental research?

DRAXIN, also known as dorsal repulsive axon guidance protein or neural tissue-specific cysteine-rich protein, is a secreted glycoprotein primarily involved in axon guidance and brain development. The human canonical protein consists of 349 amino acid residues with a molecular weight of approximately 38.7 kDa, though it often appears at ~50-68 kDa in experimental applications due to post-translational modifications, particularly glycosylation .

DRAXIN is significant in neurodevelopmental research because:

• It functions as a repulsive guidance protein for spinal cord and forebrain commissures

• It plays critical roles in the development of commissural axons during brain development

• It may act as an antagonist of canonical Wnt signaling by inhibiting β-catenin stabilization

• DRAXIN gene orthologs have been identified across multiple species including mouse, rat, bovine, frog, zebrafish, chimpanzee, and chicken

Which applications are most common for DRAXIN antibodies?

DRAXIN antibodies are utilized across multiple immunodetection methods with varying degrees of success. Based on available data, the most common applications include:

Most commercially available DRAXIN antibodies show reactivity across human, mouse, and rat species, with some also reactive to chicken DRAXIN .

What are the optimal fixation and blocking conditions for DRAXIN immunohistochemistry?

For optimal immunohistochemistry results with DRAXIN antibodies:

• Fixation: Standard fixation with 4% paraformaldehyde is generally effective. For tissues with high endogenous peroxidase activity, treatment with hydrogen peroxide (H₂O₂) is recommended before blocking .

• Blocking: Optimal blocking involves:

  • 10% BSA solution is commonly used to prevent non-specific binding

  • Incubation time of 1-2 hours at room temperature is typically sufficient

  • For tissues with high background, adding 0.1-0.3% Triton X-100 to the blocking solution can improve antibody penetration

• Antibody Dilution: Primary DRAXIN antibodies are typically used at dilutions of 1:200 to 1:500, but this may vary by manufacturer. For example, the R&D Systems AF6148 antibody has been successfully used at 10-15 μg/mL for immunofluorescence and IHC applications .

• Incubation Conditions: Overnight incubation at 4°C with the primary antibody typically yields the best signal-to-noise ratio .

How can I distinguish between DRAXIN isoforms in my experimental system?

At least three isoforms of DRAXIN have been identified, adding complexity to experimental detection. To distinguish between DRAXIN isoforms:

• Select antibodies strategically: Some antibodies, like the one described in result , detect only the two shorter isoforms. Check antibody epitope information - C-terminal antibodies may detect different isoforms than N-terminal ones .

• Use isoform-specific RT-PCR: Design primers that specifically amplify individual isoforms based on unique exon junctions.

• Employ multi-antibody approach: Use multiple antibodies targeting different epitopes in parallel experiments to identify isoform patterns.

• Western blot analysis: Different isoforms appear at distinct molecular weights:

  • The canonical isoform appears at ~38.7 kDa (unmodified)

  • Post-translationally modified forms typically appear at ~50-58 kDa

  • Some experimental systems show bands at ~68 kDa

When analyzing results, remember that differences in glycosylation patterns across cell types can affect apparent molecular weights even for the same isoform.

What are the critical considerations when using DRAXIN antibodies for analyzing neurodevelopmental processes?

When studying DRAXIN in neurodevelopmental contexts, researchers should consider:

• Developmental timing: DRAXIN expression varies significantly throughout development. Studies in mouse embryos show peak expression at specific developmental stages (E13.5 is often used for studies), while expression levels decrease in adult brain tissue .

• Regional specificity: DRAXIN shows heterogeneous expression across brain regions. Particularly high expression is observed in:

  • Diencephalon

  • Spinal cord commissural neurons

  • Forebrain commissures

• Functional redundancy: When designing knockdown/knockout experiments, consider potential compensatory mechanisms from other axon guidance molecules.

• In vivo vs. in vitro differences: DRAXIN's effects may differ between in vitro cell culture systems and in vivo models, possibly due to:

  • Differences in post-translational modifications

  • Presence/absence of binding partners

  • Three-dimensional tissue architecture effects

• Transmembrane vs. secreted forms: Studies have shown that the transmembrane form of DRAXIN has stronger functional effects than the secreted form in axonal projection experiments. This may impact the interpretation of overexpression or knockdown studies .

What troubleshooting approaches can resolve inconsistent DRAXIN antibody staining patterns?

Inconsistent DRAXIN antibody staining is a common challenge. Consider these troubleshooting approaches:

• Antibody validation: Confirm specificity through:

  • Positive controls (SH-SY5Y neuroblastoma cell line shows reliable DRAXIN expression)

  • Negative controls (absence of primary antibody)

  • siRNA knockdown validation (reduction of signal in knockdown cells)

• Post-translational modification effects: DRAXIN glycosylation can mask epitopes. Try:

  • Deglycosylation treatment before immunodetection

  • Testing multiple antibodies targeting different epitopes

  • Using reducing vs. non-reducing conditions for Western blot

• Fixation optimization: Different fixatives might reveal distinct conformational epitopes:

  • Try 4% PFA, methanol, or acetone fixation

  • Adjust fixation times (over-fixation can mask epitopes)

  • Consider antigen retrieval methods for fixed tissues

• Detection system selection: For tissues with low DRAXIN expression, signal amplification may be necessary:

  • Tyramine signal amplification

  • Use of more sensitive detection systems (e.g., HRP-DAB for IHC)

How can DRAXIN antibodies be used to assess glioma progression and prognosis?

Recent research has identified DRAXIN as a potential prognostic marker in glioma. When designing studies to investigate this relationship:

• Expression analysis approach: Analysis of multiple datasets (including CGGA, GSE50161, and laboratory samples) shows that DRAXIN is significantly upregulated in glioma tissues compared to normal brain tissue, with the highest expression in Grade IV glioma (GBM) .

• Prognostic correlation methodology:

  • High DRAXIN expression correlates with shorter survival time and poor prognosis

  • DRAXIN expression, combined with 1p19q co-deletion status and IDH mutation status, has significant prognostic value (AUC > 0.7)

  • Multivariate analysis identified DRAXIN expression as an independent risk factor for poor prognosis (HR > 1, p < 0.001)

• Technical considerations:

  • IHC protocols for DRAXIN in glioma typically use antibody dilutions of 1:500

  • Quantification of expression should be performed using standardized software (e.g., ImagePro-Plus)

  • Statistical analysis should adjust for other prognostic factors including WHO grade, PRS grade, IDH mutation, and 1p19q co-deletion status

• Functional validation: Knockdown experiments in glioma cell lines (e.g., U251) can validate DRAXIN's role in proliferation and invasion, supporting its clinical relevance. Successful knockdown typically shows:

  • ~70% reduction in DRAXIN mRNA levels

  • Significant decrease in cell proliferation (observable after 72-96 hours)

  • Reduced invasion in transwell assays

  • Decreased migration in wound healing assays

What methodological approaches can accurately distinguish DRAXIN expression in neoplastic versus normal neural tissues?

To accurately differentiate DRAXIN expression patterns between normal and neoplastic neural tissues:

• Multi-method validation:

  • Combine RT-qPCR, IHC, and Western blot analyses for comprehensive assessment

  • Use in situ hybridization (ISH) as demonstrated in the IVY-GAP database to visualize spatial expression patterns

• Reference sample selection:

  • Include appropriate normal brain tissue controls from matching brain regions

  • For developmental studies, use age-matched controls

  • Consider using both cell line models (HA cell line as normal control) and tissue samples

• Quantification strategies:

  • Employ digital image analysis with standardized parameters

  • Use H-score or similar semi-quantitative scoring systems

  • Perform blinded evaluation by multiple observers to reduce bias

• Panel approach:

  • Combine DRAXIN antibody with markers of cell proliferation (Ki67)

  • Include neural cell type-specific markers to contextualize DRAXIN expression

  • Correlate with established molecular markers (IDH status, 1p19q co-deletion)

What are the critical parameters for successful DRAXIN antibody western blotting?

To optimize Western blot detection of DRAXIN:

• Sample preparation:

  • Cell lysates: SH-SY5Y human neuroblastoma cell line is recommended as a positive control

  • Tissue samples: Embryonic brain tissues (E9-E13.5) typically show higher expression than adult brain

  • Lysis buffers containing protease inhibitors are essential to prevent degradation

• Technical parameters:

  • Molecular weight range: Monitor 38-70 kDa range

    • Unmodified DRAXIN appears at ~38.7 kDa

    • Glycosylated forms appear at ~50-58 kDa

    • Some experimental systems show bands at ~68 kDa

  • Reducing conditions: Use reducing conditions with β-mercaptoethanol

  • Transfer conditions: Semi-dry transfer at 15V for 30 minutes has been effective

  • Blocking: 5% non-fat milk or 3-5% BSA in TBST for 1 hour at room temperature

  • Primary antibody: Typically 1:500-1:1000 dilution (or 1 μg/mL for concentration-defined antibodies)

• Detection optimization:

  • Enhanced chemiluminescence (ECL) systems are generally sufficient

  • For weaker signals, consider higher sensitivity ECL substrates or longer exposure times

  • Fluorescence-based detection can provide better quantification

• Positive controls:

  • Recombinant DRAXIN protein

  • SH-SY5Y cell lysate

  • E13.5 mouse brain lysate

How can I optimize DRAXIN antibody-based immunofluorescence for studying axonal guidance mechanisms?

For studying axonal guidance using DRAXIN immunofluorescence:

• Specimen preparation:

  • For tissue sections: 10-20 μm frozen sections are preferable to paraffin sections

  • For neuronal cultures: Fixation with 4% PFA for 15-20 minutes at room temperature

  • Permeabilization with 0.1-0.3% Triton X-100 for 10-15 minutes

• Co-staining strategy:

  • Combine DRAXIN antibody with neuronal markers:

    • 23C10 or Tuj-1 for general neuronal detection

    • Specific markers for commissural neurons when relevant

  • Use different fluorophores with minimal spectral overlap (e.g., NorthernLights 557 for DRAXIN)

• Visualization approach:

  • Confocal microscopy is recommended for detailed co-localization studies

  • For axonal tracing studies, consider combining with anterograde or retrograde tracers

  • For quantitative analysis, use z-stack acquisition and 3D reconstruction

• Controls and validation:

  • Include no-primary antibody controls

  • Validate specificity using siRNA knockdown samples

  • Consider DRAXIN overexpression controls (both secreted and transmembrane forms)

• Quantification methods:

  • Measure axon length, branching patterns, or turning angles

  • Assess DRAXIN co-localization with guidance cue receptors

  • For in vivo studies, quantify abnormal axon projections as demonstrated in the chick hindbrain model (abnormal axon counts were 0.37±0.05 in control, 8.2±0.17 in secreted DRAXIN overexpression, and 27.3±4.9 in transmembrane DRAXIN overexpression groups)

How should functional studies be designed to investigate DRAXIN's role in commissural axon development?

When investigating DRAXIN's role in commissural axon development:

• Model system selection:

  • Chick embryo electroporation at HH stages 13-14 allows efficient in vivo manipulation

  • Mouse embryonic neuronal cultures provide controlled in vitro conditions

  • DRAXIN knockout mice show complete absence of all forebrain commissures

• Manipulation approaches:

  • Overexpression studies: Compare effects of:

    • Secreted DRAXIN

    • Transmembrane DRAXIN (shows stronger effects on axonal projection)

    • Empty vector control

  • Loss-of-function studies:

    • siRNA knockdown

    • CRISPR/Cas9 genome editing

    • Function-blocking antibodies

• Readout methods:

  • Whole-mount immunohistochemistry: Use 23C10 or Tuj-1 antibodies to visualize axonal projections

  • Quantification: Count abnormal projecting axons in the dorsal roof of the hindbrain

  • Section analysis: Combine with EGFP co-electroporation to identify the manipulated side

• Key experimental controls:

  • Non-targeting siRNA or empty vector controls

  • Rescue experiments to confirm specificity

  • Side-to-side comparisons within the same embryo (electroporated vs. non-electroporated side)

What are the critical considerations when using DRAXIN antibodies for investigating protein-protein interactions?

When studying DRAXIN's protein interactions:

• Co-immunoprecipitation (Co-IP) considerations:

  • Select antibodies that don't interfere with protein-protein interaction domains

  • C-terminal antibodies may be preferable as DRAXIN interacts with many partners through its N-terminal domains

  • Mild lysis conditions (e.g., NP-40 or Triton X-100 buffers) help preserve protein complexes

• Interaction partners to consider:

  • Components of the Wnt signaling pathway (β-catenin)

  • Other axon guidance molecules (particularly those involved in commissural axon development)

  • Cell surface receptors mediating DRAXIN's repulsive effects

• Proximity ligation assay (PLA) approach:

  • Allows visualization of protein interactions in situ

  • Combine DRAXIN antibody with antibodies against potential interaction partners

  • Particularly useful for cell type-specific interaction studies in heterogeneous tissues

• Cross-linking considerations:

  • For transient interactions, consider chemical cross-linking before immunoprecipitation

  • Formaldehyde (0.1-1%) or DSS (disuccinimidyl suberate) at 1-2 mM can stabilize complexes

  • Optimize cross-linking conditions to avoid epitope masking