VASN Antibody, HRP conjugated

What is Vasorin (VASN) and what role does it play in cellular signaling pathways?

Vasorin (VASN) is a transmembrane glycoprotein that plays significant roles in multiple cellular signaling pathways. Research has demonstrated that VASN interacts directly with Notch1, regulating its turnover and stability at the cell membrane. VASN binds to and stabilizes Notch1 by blocking Numb-mediated ubiquitylation and lysosomal degradation . This interaction is particularly important in pathological contexts such as glioblastoma stem cells (GSCs), where silencing Vasorin reduced Notch signaling and improved survival in mouse models .

In addition, VASN is differentially induced under hypoxic conditions through a HIF1α/STAT3 co-activator complex mechanism . Its expression correlates with multiple hypoxia response genes, including CA9, VEGFA, LDHA, PDK1, and PGK1, suggesting an important role in cellular adaptation to low oxygen environments .

What are the key advantages of using HRP-conjugated antibodies for VASN detection?

HRP (Horseradish Peroxidase)-conjugated antibodies offer several significant advantages for VASN detection:

• Enhanced signal amplification: HRP-conjugated antibodies amplify the signal of primary antibodies, enabling the detection of low-abundance proteins like VASN .

• Rapid and intense signal generation: HRP, a glycoprotein enzyme with a mass of approximately 44 kDa, catalyzes reactions that produce rapid and intense signals, making it ideal for quantitative analysis .

• Versatile substrate compatibility: HRP can be used with various substrates, including tetramethylbenzidine (TMB), diaminobenzidine (DAB), ABTS, and chemiluminescent substrates like luminol .

• Superior physical properties: Compared to alternatives like alkaline phosphatase, HRP is smaller (44 kDa vs. 140 kDa), less expensive, produces faster reactions, and exhibits greater stability in phosphate-based buffers .

• Compatibility with multiple detection methods: HRP-conjugated antibodies work effectively in Western blotting, ELISA, immunohistochemistry, and flow cytometry applications .

What are the optimal protocols for using HRP-conjugated VASN antibodies in Western blot applications?

When using HRP-conjugated VASN antibodies for Western blotting, follow these optimized protocols:

Sample preparation and incubation:

• Prepare lysates from your samples (e.g., cell/tissue extracts containing VASN)

• Separate proteins via SDS-PAGE and transfer to a membrane (nitrocellulose or PVDF)

• Block the membrane with appropriate blocking buffer (typically 5% non-fat milk or BSA)

• Incubate with primary antibodies overnight at 4°C

• Incubate with HRP-conjugated secondary antibodies (typically at 1:1000-1:5000 dilution)

Detection optimization:

• For VASN detection, use chemiluminescent substrates like luminol for higher sensitivity

• The optimal dilution should be experimentally determined, but 1:1000 is recommended as a starting point

• HRP-conjugated antibodies are optimally stored at 2-8°C and should not be frozen

• The catalytic activity of HRP can be inhibited by cyanides, azides, and sulfides, which should be avoided in buffers

Example protocol from published research:
"Blots were incubated with primary antibodies overnight at 4°C followed by HRP-conjugated species-specific antibodies (1:5000). All immunoblots were visualized using an ECL Reagent."

How can I troubleshoot non-specific binding when using HRP-conjugated VASN antibodies?

Non-specific binding is a common challenge when using HRP-conjugated antibodies for VASN detection. Here are methodological approaches to troubleshoot:

Common causes and solutions for non-specific binding:

Advanced troubleshooting approach:
For particularly challenging samples, consider implementing the Catalyzed Signal Amplification (CSA) method, also known as Tyramide Signal Amplification. This method can significantly increase sensitivity when VASN is present in very low quantities .

What controls should be included when using HRP-conjugated VASN antibodies for immunohistochemistry?

Proper controls are essential for reliable immunohistochemistry results with HRP-conjugated VASN antibodies:

Essential controls:

• Positive control: Include tissue samples known to express VASN (based on published research, hypoxic tissues often show increased VASN expression) .

• Negative control:

  • Primary antibody omission: Replace primary antibody with buffer

  • Isotype control: Use non-specific antibody of the same isotype

  • Pre-absorption control: Pre-incubate primary antibody with excess VASN antigen

• Technical controls:

  • Endogenous peroxidase control: Include steps to block endogenous peroxidase activity

  • Species cross-reactivity control: When working with wildlife or uncommon species, evaluate species-specific conjugate reactivity

Example from literature:
"The preabsorption of the conjugate with substance P obliterated the reaction," demonstrating specificity of the antibody interaction . Similarly, for VASN antibodies, pre-absorption with recombinant VASN protein should eliminate specific staining.

How can I detect VASN in extracellular vesicles using HRP-conjugated antibodies?

Detecting VASN in extracellular vesicles (EVs) requires specialized techniques due to the small size and heterogeneity of EVs:

Methodological approach:

• EV isolation:

  • Use ultracentrifugation, size exclusion chromatography, or commercial EV isolation kits

  • Verify EV purity through particle analysis (e.g., nanoparticle tracking analysis)

• VASN detection in EVs:

  • Western blot: Lyse EVs in RIPA buffer and proceed with standard Western blot protocols

  • ELISA: Immobilize EVs on plates coated with EV-capturing antibodies before VASN detection

• Special considerations for EV-associated VASN:

  • Research indicates VASN is enriched in EVs compared to source cells

  • When NP (nucleus pulposus) cells were incubated with MSC-derived EVs, "the levels of Vasorin in NP cells increased with the concentration of added EVs"

  • "Vasorin was rich in EVs fraction"

Experimental evidence:
Studies have shown that "Vasorin delivered by EVs activated Notch1 signaling and mediated therapeutic effects on NP cells" . This suggests VASN maintains its biological activity when transported via EVs, making it an important target for detection.

What is the relationship between hypoxia, HIF1α/STAT3, and VASN expression?

The relationship between hypoxia, HIF1α/STAT3, and VASN expression represents a complex regulatory network:

Hypoxic regulation of VASN:

• Induction mechanism:

  • VASN is differentially induced under hypoxic conditions

  • Requires formation of a HIF1α/STAT3 co-activator complex

  • HIF1α, but not HIF2α, is essential for VASN induction

• Experimental evidence:

  • "Under hypoxic conditions, downregulation of HIF1α but not HIF2α diminished Vasorin expression in GSCs at the protein and mRNA levels"

  • "STAT3 knockdown reduced HIF1α binding to the Vasorin promoter"

  • ChIP assays confirmed both HIF1α and STAT3 bound to the Vasorin promoter

• Regulatory pathway:

  • HIF1α is induced in both GSCs and non-GSCs under hypoxia

  • STAT3 is constitutively activated in GSCs

  • The selective expression of VASN in hypoxic GSCs requires both STAT3 and HIF1α

Correlation with hypoxia markers:
VASN expression correlates with multiple hypoxia response genes, including CA9, VEGFA, LDHA, PDK1, and PGK1 in the TCGA glioma database . This correlation supports the role of VASN as a hypoxia-responsive gene.

How does VASN regulate Notch1 signaling and what are the implications for experimental design?

VASN plays a critical role in regulating Notch1 signaling through a specific mechanism:

Mechanistic relationship:

• Stabilization mechanism:

  • VASN binds to and stabilizes Notch1 at the cell membrane

  • Blocks Numb-mediated ubiquitylation and lysosomal degradation of Notch1

  • Regulates Notch stability before proteolytic processing

• Direct interaction evidence:

  • "The immunoprecipitation revealed the integration of vasorin and Notch1"

  • "The results based on immunofluorescence analysis showed the colocalization of Vasorin and Notch1 in NP cells"

• Functional consequences:

  • Silencing Vasorin reduces Notch signaling

  • Affects downstream Notch targets (Hey1 and Hey2)

  • Reduces cell proliferation and migration dependent on Notch signaling

Experimental design implications:

"When NP cells were incubated with EVs-si-VASN (EVs with significantly decreased expression of Vasorin), the therapeutic effect mediated by EVs was mostly abrogated" , demonstrating the specific requirement for VASN in these effects.

What methods exist for producing recombinant HRP-conjugated antibodies against VASN?

Production of recombinant HRP-conjugated antibodies represents an advanced approach with specific advantages over chemical conjugation:

Recombinant production methods:

• Expression system selection:

  • Pichia pastoris methylotrophic yeast expression system has been successfully used for HRP-antibody conjugates

  • This system allows secreted production of functional conjugates

  • "The recent advance in the functional expression of HRP and antibodies in secreted form paves the way for the construction of recombinant HRP–antibody conjugates"

• Genetic construct design:

  • Create fusion proteins where HRP is linked to antibody fragments

  • Two approaches: HRP linked to N-terminal or C-terminal regions of antibody

  • Use short linker sequences like (Gly₄Ser)₃ between HRP and antibody components

• Purification and characterization:

  • Typical yield: 3-10 mg per 1L of culture supernatant

  • Chromatography-based purification

  • Characterize by SDS-PAGE, Western blot, and functional assays

Advantages of recombinant conjugates:
"Recombinant immunoconjugates of marker enzymes with antigens or antibodies present considerably more advantages than those obtained by conventional methods of chemical synthesis; i.e., they are homogeneous, have a strictly determined stoichiometry, and retain the functional activity of both a marker protein and an antigen/antibody" .

How can I optimize Western blot protocols specifically for detecting low levels of VASN?

When VASN is present at low levels, standard Western blot protocols may be insufficient. Here are optimized approaches:

Enhanced detection strategies:

• Sample preparation optimization:

  • Enrich for membrane fractions (VASN is a transmembrane protein)

  • Use phosphatase inhibitors (as phosphorylation may affect detection)

  • Consider immunoprecipitation to concentrate VASN before Western blotting

• Signal amplification methods:

  • Implement Catalyzed Signal Amplification (CSA) method for enhanced sensitivity

  • "This method is not routinely used in IHC but might be useful when the antigen of interest is present in very low quantity"

  • Use high-sensitivity chemiluminescent substrates like Radiance Q or Radiance ECL

• Incubation conditions optimization:

  • Extended primary antibody incubation (overnight at 4°C)

  • Optimize secondary antibody dilution (typically 1:1000 to 1:5000)

  • Add 0.1% Triton X-100 to improve antibody penetration

Substrate selection considerations:
For optimal detection of low-abundance VASN, choose substrates based on required sensitivity:

• For extreme sensitivity: Chemiluminescent substrates like luminol

• For colorimetric detection: DAB provides good sensitivity and stable signal

• For possible multiplex detection: Consider fluorogenic HRP substrates

What are the cross-species reactivity considerations when using VASN antibodies in different model organisms?

Cross-species reactivity is a critical consideration when developing or selecting VASN antibodies for use across different model organisms:

Cross-reactivity considerations:

• Species-specific conjugate development:

  • Commercial conjugates like protein-G and protein-AG demonstrate variable and often low relative avidity (<20%) against many wildlife species

  • Species-specific conjugates typically show higher avidity (>70%) against target species

  • Cross-reactivity can be systematically evaluated using ELISA-based avidity indices

• Empirical cross-reactivity data:

  • Research has shown that "commercially available protein-G and protein-AG demonstrate significantly low relative avidity (<20%) against many species"

  • Species-specific conjugates demonstrated cross-reactivity with a mean relative avidity of >50% with 18 other wildlife species

• Validation requirements:

  • Western blot with positive control samples from each species

  • Immunohistochemistry with known VASN-expressing tissues

  • Competitive binding assays to confirm specificity

Practical approach for model organism work:
"These results demonstrate that species-specific conjugates are important tools for the development and validation of immunoassays in wildlife and for the surveillance of zoonotic agents along the livestock-wildlife-human interface" . When working with non-standard model organisms, consider developing species-specific secondary antibodies or validating commercial options with appropriate controls.

How do HRP-conjugated antibodies compare with other detection systems for VASN visualization?

Different detection systems offer various advantages for VASN visualization in different experimental contexts:

Comparison of detection systems:

Comparative performance:
"Compared to another common enzyme, alkaline phosphatase, HRP is known to be smaller in size, less expensive, produce faster reactions, and exhibit greater stability, particularly in the presence of phosphate-based buffers" .

For VASN detection specifically, HRP conjugates provide optimal sensitivity for detecting this protein in contexts such as hypoxic tissues and extracellular vesicles, where expression levels may vary significantly.

What methods can be used to create multiplex detection systems involving VASN and interacting proteins like Notch1?

Multiplex detection of VASN and its interacting partners requires specialized approaches:

Multiplex detection strategies:

• Multiple chromogenic substrates:

  • Use different enzymes (HRP and AP) with contrasting substrates

  • "HRP can be used with a wide range of substrates, such as tetramethylbenzidine (TMB), diaminobenzidine (DAB), ABTS, chemiluminescent substrates like luminol, and some fluorogenic substrates"

  • Example: VASN detected with HRP-DAB (brown) and Notch1 with AP-Fast Blue (blue)

• Immunofluorescence multiplex:

  • Use different fluorophores for each target

  • Employ sequential staining if using same-species antibodies

  • Has been successfully used to show "the colocalization of Vasorin and Notch1 in NP cells"

• Proximity ligation assay:

  • Detect protein-protein interactions in situ

  • Particularly valuable for confirming VASN-Notch1 interactions

  • Generate fluorescent signal only when proteins are in close proximity

Sequential immunoprecipitation approach:
"The immunoprecipitation revealed the integration of vasorin and Notch1" . This technique can be expanded by:

• Immunoprecipitate with anti-VASN

• Probe with anti-Notch1

• Strip and re-probe for other interacting proteins