VASN Antibody

What is VASN and why is it important in biological research?

VASN (vasorin) is a type I membrane protein with 673 amino acid residues and a mass of 71.7 kDa in humans. It functions primarily as an inhibitor of TGF-beta signaling and is predominantly expressed in vascular smooth muscle cells in a developmentally regulated pattern. Research interest in VASN stems from its role in vessel repair after arterial injury, where its expression is significantly down-regulated. VASN directly binds to transforming growth factor (TGF)-beta and attenuates TGF-beta signaling, making it a critical protein for studies investigating vascular development, repair processes, and TGF-beta pathway regulation .

Which applications are most common for VASN antibodies in research?

VASN antibodies are utilized across multiple immunological detection methods, with varying effectiveness depending on the experimental context. The most widely employed applications include:

When designing experiments, researchers should consider that VASN antibodies demonstrate highest reactivity in aortic tissue, intermediate reactivity in kidney and placenta, and lowest reactivity in brain, heart, liver, lung and skeletal muscle tissues .

How should researchers properly validate a VASN antibody before experimental use?

Proper antibody validation should follow a multi-step process to ensure specificity and reproducibility:

• Positive control verification: Test the antibody on tissues/cells known to express VASN abundantly (e.g., aortic tissue, vascular smooth muscle cells).

• Negative control testing: Confirm minimal reactivity in tissues with low VASN expression (e.g., brain tissue).

• Molecular weight confirmation: In Western blot applications, verify detection of specific bands at approximately 65 and 95 kDa (the latter representing glycosylated forms).

• Cross-reactivity assessment: If working with non-human models, confirm reactivity with the species-specific VASN protein (considering orthologs in mouse, rat, bovine, etc.).

• Knockdown/knockout validation: When possible, confirm specificity using VASN-knockdown or knockout samples.

This comprehensive validation approach minimizes the risk of non-specific binding and ensures experimental reproducibility across different research contexts .

What are the optimal conditions for Western blot detection of VASN protein?

For optimal Western blot detection of VASN, researchers should implement the following protocol parameters:

• Sample preparation: Use RIPA buffer supplemented with protease inhibitors for tissue or cell lysis.

• Reducing conditions: Always run samples under reducing conditions as the antibody recognition may be affected by protein conformational changes.

• Antibody concentration: Use 2 μg/mL of anti-VASN antibody for optimal signal-to-noise ratio.

• Membrane selection: PVDF membranes provide better results than nitrocellulose for VASN detection.

• Blocking solution: 5% non-fat dry milk in TBST performs better than BSA-based blockers.

• Expected bands: Look for specific bands at approximately 65 kDa (unmodified protein) and 95 kDa (glycosylated form).

• Secondary antibody: Use HRP-conjugated secondary antibodies for standard chemiluminescent detection.

This methodology has been validated in studies examining human placenta and lung tissue lysates, providing consistent and reproducible results .

How can researchers optimize immunohistochemical detection of VASN in tissue samples?

For optimal immunohistochemical detection of VASN in fixed tissue samples:

• Fixation method: Use 10% neutral buffered formalin; avoid Bouin's fixative which can interfere with epitope recognition.

• Antigen retrieval: Heat-induced epitope retrieval using citrate buffer (pH 6.0) for 20 minutes provides optimal results.

• Blocking procedure: Block with 10% normal serum from the species of the secondary antibody for 1 hour at room temperature.

• Primary antibody incubation: Dilute antibody 1:100-1:200 and incubate overnight at 4°C.

• Detection method: Streptavidin-biotin amplification systems provide better sensitivity than direct detection methods.

• Counterstaining: Hematoxylin counterstaining at 25% normal strength allows better visualization of positive signals.

• Controls: Always include positive controls (aorta tissue) and negative controls (antibody diluent without primary antibody).

This approach has been successfully employed in studies examining VASN expression patterns in vascular tissues and during vessel repair processes .

What methodological considerations are important when using VASN antibodies for co-immunoprecipitation studies?

When conducting co-immunoprecipitation (co-IP) studies with VASN antibodies to investigate protein-protein interactions:

• Lysis buffer selection: Use mild, non-denaturing buffers (e.g., NP-40 or Triton X-100 based) to preserve protein-protein interactions.

• Pre-clearing: Pre-clear lysates with protein A/G beads to reduce non-specific binding.

• Antibody binding: Use 5 μg of purified VASN antibody per 500 μg of total protein lysate.

• Incubation conditions: Perform the antibody-lysate incubation overnight at 4°C with gentle rotation.

• Washing stringency: Use progressively stringent wash buffers to minimize background while preserving specific interactions.

• Elution conditions: Elute under mild conditions if the goal is to maintain native protein structure.

• Verification strategy: Confirm results with reciprocal co-IP using antibodies against the interacting partner (e.g., anti-TGF-beta antibodies).

This method has been successfully employed to demonstrate VASN's direct interaction with TGF-beta, contributing to our understanding of how VASN attenuates TGF-beta signaling .

How can VASN antibodies be utilized to study the cleavage of VASN by ADAM17 (TACE) and its impact on TGF-beta signaling?

Research has revealed that ADAM17 (TACE) regulates TGF-beta signaling through the cleavage of vasorin. To investigate this regulatory mechanism:

• Experimental design: Implement a dual-detection approach using antibodies that recognize both the full-length VASN and its cleaved fragments.

• Cell models: Compare VASN cleavage patterns in ADAM17-expressing versus ADAM17-knockdown cell lines.

• Inhibitor studies: Utilize specific ADAM17 inhibitors (e.g., TAPI-2) to confirm the specificity of cleavage events.

• Downstream analysis: Use phospho-specific antibodies against SMAD proteins to quantify TGF-beta signaling activity.

• Recombinant protein studies: Perform in vitro cleavage assays with purified ADAM17 and recombinant VASN.

• Domain mapping: Use domain-specific VASN antibodies to identify precise cleavage sites.

This approach has yielded important insights into how ADAM17-mediated cleavage of VASN contributes to the regulation of TGF-beta signaling pathways, with potential implications for vascular development and disease .

What strategies can be employed to investigate VASN's role in regulating autophagy-mediated apoptosis in transformed cells?

Recent research has connected VASN/ATIA to cigarette smoke-induced transformation of human bronchial epithelial cells through suppression of autophagy-mediated apoptosis. To investigate this phenomenon:

• Cell model selection: Use paired normal and transformed epithelial cell lines with differential VASN expression levels.

• Expression manipulation: Implement VASN overexpression and knockdown approaches to establish causality.

• Autophagy markers: Quantify LC3-I to LC3-II conversion and p62 levels using Western blot analysis.

• Autophagic flux: Measure autophagic flux using bafilomycin A1 treatment combined with VASN antibody-based detection of autophagy markers.

• Apoptosis assessment: Combine annexin V staining with cleaved caspase-3/7 detection to quantify apoptotic responses.

• Pathway inhibition: Use pharmacological inhibitors of autophagy (e.g., 3-methyladenine) and apoptosis (e.g., Z-VAD-FMK) to delineate pathway interactions.

• In vivo validation: Utilize xenograft models with VASN-manipulated cell lines to confirm in vitro findings.

This comprehensive approach has revealed that VASN modulates cellular transformation by regulating the balance between autophagy and apoptosis, providing new insights into potential therapeutic targets for smoking-related malignancies .

How can researchers effectively use VASN antibodies to study its role as a biomarker in ovarian cancer?

VASN has been identified in conditioned media from ovarian cancer cell lines, suggesting potential as a biomarker. To investigate this application:

• Sample collection strategy: Collect matched serum, ascites, and tissue samples from ovarian cancer patients across different disease stages and histological subtypes.

• Detection method optimization: Develop a sandwich ELISA using capture and detection antibodies targeting different VASN epitopes.

• Sensitivity enhancement: Implement signal amplification methods to detect low VASN concentrations in early-stage disease.

• Specificity verification: Include appropriate controls (benign conditions, other cancer types) to establish diagnostic specificity.

• Comparative analysis: Compare VASN levels with established biomarkers (e.g., CA-125) through ROC curve analysis.

• Multivariate modeling: Develop predictive models incorporating VASN with other biomarkers and clinical parameters.

• Longitudinal assessment: Monitor VASN levels throughout treatment to evaluate potential as a treatment response marker.

This research strategy has identified VASN as a potential novel marker for epithelial ovarian cancer, with ongoing investigations to determine its clinical utility in early detection and treatment monitoring paradigms .

What are common causes of weak or absent signal when using VASN antibodies in Western blot applications?

When faced with weak or absent signals in Western blot experiments using VASN antibodies, consider these methodological solutions:

Additionally, researchers should note that VASN undergoes N-glycosylation, which can affect its migration pattern on SDS-PAGE gels. Treatment with glycosidases prior to Western blot can help distinguish between glycosylated and unmodified forms of the protein .

How can researchers address cross-reactivity concerns when studying VASN in various animal models?

When using VASN antibodies across different species, researchers should implement these strategies to address cross-reactivity concerns:

• Sequence homology analysis: Compare VASN amino acid sequences between human and the target species to identify conserved epitopes.

• Species validation testing: Test the antibody on positive control samples from each species before conducting main experiments.

• Epitope-specific antibody selection: Choose antibodies targeting highly conserved regions for cross-species applications.

• Antibody concentration optimization: Titrate antibody concentrations for each species separately.

• Preabsorption controls: Perform preabsorption with recombinant VASN protein to confirm specificity.

• Knockout/knockdown validation: When available, use VASN knockout/knockdown samples as negative controls.

• Alternative antibody consideration: If cross-reactivity issues persist, consider species-specific antibodies developed against the orthologous protein.

The VASN gene has orthologs in mouse, rat, bovine, frog, zebrafish, chimpanzee, and chicken, making careful antibody selection critical for comparative studies across these species .

What techniques can researchers employ to enhance detection sensitivity for low-abundance VASN in certain tissue types?

For tissues with low VASN expression (brain, heart, liver), these sensitivity enhancement strategies should be implemented:

• Sample enrichment: Perform subcellular fractionation to concentrate membrane proteins where VASN naturally localizes.

• Signal amplification systems: Use tyramide signal amplification (TSA) for immunohistochemistry/immunofluorescence applications.

• Proximity ligation assay (PLA): Implement PLA to visualize and quantify low-abundance VASN protein interactions.

• Extended antibody incubation: Increase primary antibody incubation time to 48-72 hours at 4°C for enhanced epitope binding.

• High-sensitivity detection reagents: Use femto-level chemiluminescent substrates for Western blot applications.

• Antibody concentration: Increase antibody concentration while maintaining an optimal signal-to-noise ratio.

• Reduced sample dilution: Load higher protein amounts (50-100 μg) for Western blot detection.

• Immunoprecipitation before detection: Concentrate the target protein through immunoprecipitation before Western blot analysis.

These approaches have successfully enabled detection of VASN in tissues previously thought to have minimal expression, revealing previously uncharacterized roles in non-vascular tissues .

How can VASN antibodies be employed in single-cell analysis techniques to investigate heterogeneous expression patterns?

To leverage VASN antibodies in cutting-edge single-cell analysis:

• Single-cell Western blot: Adapt microfluidic single-cell Western blot platforms using VASN antibodies to quantify expression variability among individual cells.

• Mass cytometry (CyTOF): Conjugate VASN antibodies with metal isotopes for high-dimensional analysis of protein expression at single-cell resolution.

• Imaging mass cytometry: Combine VASN antibody detection with spatial information to map expression patterns within tissue architecture.

• Proximity extension assay: Develop VASN-specific oligonucleotide-conjugated antibody pairs for high-sensitivity protein quantification in minute samples.

• CODEX multiplexed imaging: Incorporate VASN antibodies into multiplexed imaging panels to visualize co-expression with other proteins in individual cells.

• scRNA-seq validation: Use VASN antibodies to validate protein-level expression of transcriptional findings from single-cell RNA sequencing.

These advanced approaches allow researchers to move beyond bulk tissue analysis to understand cell-specific VASN expression patterns and their functional implications in heterogeneous tissues like vascular walls and tumors .

What methodological approaches can researchers use to study the dynamic regulation of VASN in response to vascular injury?

To investigate the dynamic regulation of VASN during vascular injury and repair:

• In vivo injury models: Implement balloon injury or wire injury models in animal vasculature followed by temporal sampling.

• Ex vivo vessel culture: Establish precision-cut vessel slice cultures to monitor VASN expression changes under controlled conditions.

• Live-cell imaging: Develop fluorescently tagged VASN constructs for real-time visualization of protein dynamics.

• Inducible expression systems: Create tetracycline-regulated VASN expression systems to study the effects of controlled VASN modulation.

• Quantitative immunohistochemistry: Use digital pathology tools to quantify VASN expression levels across vessel wall layers at different timepoints after injury.

• Protein turnover analysis: Implement pulse-chase experiments with metabolic labeling to measure VASN protein half-life during injury response.

• Chromatin immunoprecipitation: Identify transcription factors regulating VASN expression during vascular repair.

This methodology has revealed that VASN expression is significantly down-regulated during vessel repair after arterial injury, suggesting a critical role in vascular remodeling processes .

How can researchers effectively investigate the interplay between VASN and TGF-beta signaling in pathological conditions?

To unravel the complex relationship between VASN and TGF-beta signaling in disease contexts:

• Dual immunostaining: Perform co-localization studies using VASN and TGF-beta pathway component antibodies in diseased tissues.

• Receptor competition assays: Develop quantitative binding assays to measure how VASN affects TGF-beta/receptor interactions.

• Phospho-SMAD profiling: Quantify SMAD phosphorylation dynamics in response to VASN manipulation using phospho-specific antibodies.

• CRISPR-engineered cellular models: Create precise VASN domain mutants to map regions critical for TGF-beta interaction.

• Transgenic animal models: Develop tissue-specific VASN knockout animals to study pathway dysregulation in vivo.

• Patient-derived samples: Compare VASN/TGF-beta pathway activation in matched normal and diseased human samples.

• Therapeutic modulation: Test the effects of TGF-beta pathway inhibitors on VASN expression and vice versa.

• Multi-omics integration: Combine proteomics, transcriptomics, and phospho-proteomics to build comprehensive signaling network models.

This integrated approach has revealed that VASN directly binds to TGF-beta and attenuates TGF-beta signaling, with ADAM17-mediated cleavage of VASN serving as a key regulatory mechanism in this pathway .