ITGAV Antibody, Biotin conjugated

What is ITGAV and what role does it play in cellular function?

ITGAV (Integrin alpha V), also known as CD51, is a critical subunit of integrin heterodimers that plays essential roles in development through cell adhesion and signaling pathways. Integrin αV-containing heterodimers are fundamental to numerous biological processes including cell migration, adhesion, and phagocytosis. ITGAV has been shown to interact with extracellular matrix proteins and participates in crucial immunomodulatory signaling pathways . The protein has significant functions in TGF-β activation and signaling, which explains why defects in ITGAV-null mice phenocopy observations in patients with loss-of-function variants in TGFB1, including brain defects and colitis . Understanding ITGAV's structure and function is crucial for interpreting experimental results when using ITGAV antibodies in research applications.

What is the molecular structure of ITGAV and how does biotin conjugation affect antibody function?

ITGAV has a calculated molecular weight of 114.6 kDa, though it migrates as approximately 135 kDa under non-reducing conditions due to glycosylation . When forming a heterodimer with ITGB3, the complex creates the biologically active αVβ3 integrin. Biotin conjugation to ITGAV antibodies is achieved through affinity chromatography under optimal conditions to ensure the solution is free of unconjugated biotin . This conjugation enables versatile detection methods through the strong biotin-streptavidin interaction without significantly affecting the antibody's antigen recognition properties. The biotin tag provides amplification opportunities in detection systems, which is particularly valuable when studying proteins with lower expression levels or in complex tissue environments.

How do ITGAV antibodies compare in specificity across different host species?

ITGAV antibodies are produced in various host species, with mouse monoclonal and rabbit polyclonal being the most common . Mouse-derived monoclonal antibodies like the NKI-M9 clone offer high specificity for human ITGAV and are particularly useful for flow cytometry applications . Rabbit polyclonal antibodies provide broader epitope recognition and are effective across multiple applications including Western blotting, immunohistochemistry, and immunofluorescence, with some showing cross-reactivity to mouse and rat ITGAV . When selecting an antibody for your research, consider the experimental technique, required sensitivity, and the species being studied to determine whether a monoclonal or polyclonal approach would be more appropriate.

How can ITGAV antibodies be used to investigate immune dysregulation mechanisms?

ITGAV plays a significant role in immune regulation, with research showing that αVβ3 downregulates Toll-like receptor (TLR) signaling in B cells by directing ligated TLRs to degradative compartments . This mechanism is central to preventing autoimmunity, as ITGAV-deficient lymphocytes demonstrate increased and prolonged TLR signaling, enhanced B cell activation, and elevated antibody production . When designing experiments to investigate these pathways, use biotin-conjugated ITGAV antibodies in flow cytometry to identify and isolate specific immune cell populations. Combine this with functional assays measuring TLR signaling output to assess the impact of ITGAV modulation. This approach enables researchers to correlate ITGAV expression levels with immune cell function and can reveal mechanisms underlying immune dysregulation in disease models.

What methodologies are effective for studying ITGAV's role in TGF-β signaling pathways?

To investigate ITGAV's role in TGF-β signaling, researchers should employ a multi-faceted approach combining protein-protein interaction studies, signaling pathway analysis, and functional outcomes. First, use biotin-conjugated ITGAV antibodies for co-immunoprecipitation experiments to identify binding partners in the TGF-β pathway. Follow with phosphorylation studies of downstream SMAD proteins, particularly SMAD3, as reduced SMAD3 expression has been observed in models with ITGAV deficiency . For robust pathway analysis, combine these protein studies with RNA sequencing to identify transcriptional changes. The zebrafish model has proven valuable for studying ITGAV function, as genetic deletion of itgav in zebrafish recapitulates patient phenotypes including retinal and brain defects, microglia loss, and colitis with reduced SMAD3 expression . This comprehensive approach provides mechanistic insights into how ITGAV regulates TGF-β signaling in different cellular contexts.

How can ITGAV antibodies be utilized in studying neurodevelopmental and inflammatory disorders?

Biallelic variants in the ITGAV gene have been associated with a spectrum of disorders including eye and brain abnormalities, inflammatory bowel disease, and other developmental issues . When investigating these conditions, implement a research strategy that examines both tissue-specific expression patterns and functional outcomes. For neurodevelopmental studies, use biotin-conjugated ITGAV antibodies in immunohistochemistry or immunofluorescence to map ITGAV expression in brain tissues across developmental stages. In inflammatory bowel disease models, combine flow cytometry with ITGAV antibodies to profile immune cell infiltrates in gut tissue, and correlate with cytokine production measurements. Patient-derived cells offer a valuable resource for functional studies, allowing researchers to establish direct links between genetic variants and cellular phenotypes . This translational approach bridges basic research findings with clinical manifestations.

What are the optimal conditions for using biotin-conjugated ITGAV antibodies in flow cytometry?

For flow cytometry applications using biotin-conjugated ITGAV antibodies, begin with careful sample preparation to ensure single-cell suspensions with minimal cell clumping or death. Optimal staining typically employs antibody dilutions between 1:100 to 1:500, though this should be empirically determined for each lot . Include proper blocking steps with 1-2% BSA or serum matched to the secondary detection reagent's host to minimize non-specific binding. For detection, use fluorophore-conjugated streptavidin (commonly PE, APC, or fluorescein), adding it in a separate incubation step after washing away excess primary antibody. Include appropriate compensation controls when using multiple fluorophores and implement FMO (fluorescence minus one) controls to accurately set gates. For multiparameter analysis, consider using the NKI-M9 clone which has been validated for human ITGAV detection in flow cytometry . This approach enables precise identification and quantification of ITGAV-expressing cell populations.

What techniques ensure successful immunoprecipitation using biotin-conjugated ITGAV antibodies?

For successful immunoprecipitation with biotin-conjugated ITGAV antibodies, implement a protocol that leverages the strong biotin-streptavidin interaction while preserving protein complexes. Begin with careful cell lysis using a buffer containing 1% NP-40 or Triton X-100, 150mM NaCl, 50mM Tris pH 7.4, and protease/phosphatase inhibitors. Pre-clear lysates with streptavidin beads to reduce non-specific binding. Incubate cleared lysates with biotin-conjugated ITGAV antibody (typically 2-5 μg per mg of total protein) overnight at 4°C with gentle rotation. Capture antibody-antigen complexes using streptavidin-coated magnetic beads for 1-2 hours, followed by 4-5 washes with decreasing salt concentration. When studying ITGAV interactions with TGF-β pathway components or other binding partners, consider using chemical crosslinking before lysis to stabilize transient interactions. This approach is particularly valuable for capturing the ITGAV-ITGB3 heterodimer, which has a calculated MW of 114.6 kDa (ITGAV) and 81.8 kDa (ITGB3) .

How can biotin-conjugated ITGAV antibodies be optimized for ELISA applications?

To optimize ELISA protocols using biotin-conjugated ITGAV antibodies, first determine whether a direct, indirect, sandwich, or competitive format is most appropriate for your research question. For sandwich ELISA detecting ITGAV in samples, coat plates with a capture antibody recognizing a different ITGAV epitope than your biotin-conjugated detection antibody. When using commercial antibodies, begin with the manufacturer's recommended dilution (typically 1:500-1:5000 for polyclonal antibodies) , then perform a titration to determine optimal concentration. For detection, use high-sensitivity streptavidin-HRP conjugates, which provide signal amplification through the biotin-streptavidin interaction. Implement rigorous washing steps (5-6 washes per stage) using PBS with 0.05% Tween-20 to minimize background. When quantifying results, prepare a standard curve using recombinant ITGAV protein with known concentration. The linear detection range for human ITGAV-ITGB3 heterodimer binding to vitronectin has been established at 0.01-1.25 μg/mL in validated systems . This methodological approach ensures sensitive and specific quantification of ITGAV in experimental samples.

How can researchers address non-specific binding issues with biotin-conjugated ITGAV antibodies?

Non-specific binding is a common challenge when working with biotin-conjugated antibodies. To minimize this issue, implement a comprehensive blocking strategy using 2-5% BSA or serum matched to the host species of your secondary detection reagent. For tissues with high endogenous biotin, pre-block with avidin/biotin blocking kits before applying the primary antibody. When experiencing high background in immunohistochemistry or immunofluorescence, adding 0.1-0.3% Triton X-100 to your blocking solution can reduce non-specific membrane interactions. Always include appropriate negative controls in your experimental design, such as isotype controls (IgG2a kappa for mouse monoclonal antibodies like NKI-M9) and secondary-only controls. If non-specific binding persists, consider further antibody titration or switching to a different clone targeting a more specific ITGAV epitope. For flow cytometry applications, proper gating strategies including FMO controls can help distinguish true signal from background fluorescence.

What approaches can resolve contradictory data when studying ITGAV in different experimental systems?

When encountering contradictory results across different experimental systems, implement a systematic troubleshooting approach. First, verify antibody specificity through validation experiments like knockdown/knockout controls or peptide competition assays. Consider that ITGAV functions may differ between cell types due to varying heterodimer partners (ITGAV can pair with β1, β3, β5, β6, and β8 subunits) and expression levels. Examine experimental conditions carefully, as ITGAV-dependent pathways are influenced by microenvironmental factors like extracellular matrix composition and TGF-β availability. When comparing in vitro findings with in vivo models, account for the complexity of tissue environments and compensatory mechanisms. The zebrafish model has proven valuable for validating human ITGAV variant effects, recapitulating patient phenotypes including retinal defects, brain abnormalities, and colitis . Integrating data from multiple methodological approaches (genetic, biochemical, and cellular) provides the most comprehensive understanding of ITGAV function in different contexts.

How should researchers interpret ITGAV expression data across different tissue and cell types?

Interpreting ITGAV expression data requires consideration of tissue-specific contexts and heterodimer formation. When analyzing immunostaining or flow cytometry results, compare ITGAV expression patterns with known binding partners like ITGB3 to determine which functional integrin complexes are likely present. Consider developmental stage effects, as ITGAV expression and function change during embryonic development and tissue maturation. This is particularly relevant when studying neurodevelopmental or retinal phenotypes associated with ITGAV variants . For quantitative comparisons across tissues, normalize ITGAV expression to appropriate housekeeping genes or proteins specific to each tissue type. When analyzing RNA-seq data, examine not only ITGAV expression but also its correlation with pathway components, particularly TGF-β signaling factors and SMAD3 . This integrated approach accounts for the complex regulatory networks influencing ITGAV function and provides more meaningful biological interpretation than examining ITGAV expression in isolation.

What new insights have emerged about ITGAV's role in immune modulation and potential therapeutic applications?

Recent research has revealed ITGAV's sophisticated role in immune regulation beyond cell adhesion. The αVβ3 integrin has been shown to downregulate Toll-like receptor signaling in B cells by targeting ligated TLRs to degradative compartments, thereby preventing excessive B cell activation and antibody production . This mechanism represents a previously unrecognized checkpoint in autoimmunity, as ITGAV-deficient lymphocytes demonstrate enhanced TLR signaling and predisposition to autoimmune conditions . Additionally, αVβ3 limits cytokine production by plasmacytoid dendritic cells and prevents activation of autoreactive B cells . These findings suggest therapeutic potential in conditions characterized by immune dysregulation, including inflammatory bowel disease, which has been observed in patients with ITGAV variants. Research combining biotin-conjugated ITGAV antibodies with functional immune assays can further elucidate these pathways and identify intervention points for modulating immune responses in disease states.

How can combined mucosal and systemic immunization approaches be optimized when studying ITGAV-related immune responses?

Studies of antibody responses in mucosal and systemic immunization models have shown significant enhancement of IgG2a responses with combined intranasal (i.n.) and intramuscular (i.m.) approaches . When designing experiments to investigate ITGAV's role in these responses, implement a protocol comparing different immunization sequences. The 2 i.n./2 i.m. immunization strategy has demonstrated significantly higher IgG2a responses compared to other approaches (p = 1.4 × 10^-5 compared to 4 i.m.) . This corresponds with enhanced splenic interferon-γ responses, indicating correlation between serum IgG2a and systemic Th1 responses . To study ITGAV's contribution to these effects, use flow cytometry with biotin-conjugated ITGAV antibodies to track integrin expression on relevant immune cell populations throughout the immunization schedule. This approach enables researchers to correlate ITGAV expression dynamics with antibody class switching and T helper cell polarization, providing mechanistic insights into how integrin signaling influences the outcome of different immunization strategies.

What experimental approaches best address the complex interplay between ITGAV and TGF-β signaling in disease models?

The interplay between ITGAV and TGF-β signaling represents a complex regulatory network with significant implications for development and disease. To effectively study this interaction, implement a multi-level experimental approach beginning with protein interaction studies using biotin-conjugated ITGAV antibodies for co-immunoprecipitation of TGF-β pathway components. Follow with phospho-specific antibody detection of SMAD proteins to assess pathway activation status. At the transcriptional level, RNA sequencing in models with ITGAV variants or knockdown can reveal global changes in TGF-β-responsive genes. The zebrafish model offers particular advantages for studying these pathways in vivo, as genetic deletion of itgav in zebrafish reproduces patient phenotypes including reduced SMAD3 expression and transcriptional dysregulation . For translational relevance, patient-derived cells can be used to validate findings from model systems, connecting genetic variants with cellular phenotypes . This comprehensive strategy provides mechanistic understanding of how ITGAV variants disrupt TGF-β signaling and contribute to conditions like inflammatory bowel disease, retinal abnormalities, and neurodevelopmental disorders.