ITGAV Antibody, HRP conjugated

What is ITGAV and what biological functions does it serve in cellular processes?

ITGAV (Integrin alpha V) is a transmembrane receptor protein that forms heterodimers with various beta subunits, most notably ITGB3, ITGB5, ITGB6, and ITGB8. These heterodimers function as receptors for a wide array of ligands including vitronectin, cytotactin, fibronectin, fibrinogen, laminin, matrix metalloproteinase-2, osteopontin, osteomodulin, prothrombin, thrombospondin, and von Willebrand factor. ITGAV-containing integrins specifically recognize and bind to the R-G-D (Arg-Gly-Asp) sequence present in numerous ligands .

The biological significance of ITGAV extends to multiple cellular processes including:

• Cell adhesion and migration

• Extracellular matrix interactions

• Signal transduction pathways

• Growth factor signaling modulation

Recent research has identified ITGAV:ITGB3 as a binding partner for various growth factors including FGF1, IGF1, IGF2, and IL1B, with this interaction being essential for their respective signaling pathways . Additionally, ITGAV:ITGB5 has been identified as a critical integrin pair necessary for cancer cell expansion, highlighting its potential role in oncogenic processes .

How does the HRP conjugation in ITGAV antibodies enhance detection capabilities compared to unconjugated antibodies?

HRP (Horseradish Peroxidase) conjugation provides significant advantages for ITGAV detection in research applications through a direct enzymatic detection system. The HRP enzyme catalyzes the oxidation of substrates (such as TMB) in the presence of hydrogen peroxide, producing a colorimetric, chemiluminescent, or fluorescent signal depending on the substrate used .

The primary methodological advantages include:

• Increased sensitivity: The enzymatic amplification allows for detection of low abundance ITGAV proteins

• Reduced protocol complexity: Direct conjugation eliminates the need for secondary antibody incubation steps

• Decreased background: Fewer antibody layers result in reduced non-specific binding

• Faster experimental timeline: Elimination of secondary antibody incubation shortens the protocol

For ITGAV detection specifically, HRP-conjugated antibodies are particularly valuable in Western blot applications (recommended dilution 1:1,000-1:2,000) and immunohistochemistry where precise localization of the protein is required . The enzymatic reaction with TMB substrate produces a measurable color change at 450nm, enabling quantitative analysis of ITGAV abundance .

What are the structural domains of ITGAV that influence antibody binding and specificity?

The ITGAV protein contains several distinct structural domains that influence antibody recognition and experimental outcomes. The most critical domain is the β-propeller domain, which contains a central pocket essential for heterodimer formation with beta integrin partners .

Recent CRISPR-tiling studies have specifically pinpointed this integral pocket within the β-propeller domain as crucial for ITGAV-ITGB5 dimerization . This structural insight is particularly important when selecting antibodies, as those targeting epitopes within or near this region may potentially interfere with natural heterodimer formation and alter experimental outcomes.

Additionally, ITGAV contains:

• A heavy chain and light chain region that form the complete integrin structure

• Extracellular domains containing ligand-binding regions that recognize RGD motifs

• Transmembrane regions anchoring the protein to the cell membrane

• Cytoplasmic domains involved in intracellular signaling

When selecting an ITGAV antibody for specific applications, researchers should consider which domain is being targeted, as this may influence protein detection in different experimental contexts or states of activation .

What are the optimal sample preparation protocols for using ITGAV Antibody, HRP conjugated in Western Blot applications?

For optimal Western blot results with HRP-conjugated ITGAV antibody, sample preparation must preserve protein integrity while maximizing epitope accessibility. The following methodological approach has been validated across multiple studies:

Sample Extraction Protocol:

• Harvest cells during logarithmic growth phase (70-80% confluence)

• Wash cells twice with ice-cold PBS to remove media components

• Lyse cells using a buffer containing:

  • 50mM Tris-HCl (pH 7.4)

  • 150mM NaCl

  • 1% NP-40 or Triton X-100

  • 0.5% sodium deoxycholate

  • Protease inhibitor cocktail

• Incubate on ice for 30 minutes with gentle agitation

• Centrifuge at 14,000×g for 15 minutes at 4°C

• Collect supernatant and determine protein concentration

SDS-PAGE and Transfer Parameters:

• Load 20-40μg of total protein per lane

• Separate proteins on 7.5-10% polyacrylamide gels (ITGAV has a calculated molecular weight of 116kDa)

• Transfer to PVDF membrane at 100V for 60-90 minutes in cold transfer buffer

• Block with 5% non-fat dry milk in TBST for 1 hour at room temperature

Antibody Incubation:

• Dilute HRP-conjugated ITGAV antibody at 1:1,000-1:2,000 in blocking buffer

• Incubate membrane overnight at 4°C with gentle agitation

• Wash membrane 3 times with TBST, 5 minutes each

• Develop using enhanced chemiluminescence substrate

This protocol has been validated with multiple cancer cell lines including MCF-7 cells, which demonstrate reliable ITGAV detection .

How should researchers optimize immunohistochemical detection of ITGAV in tissue samples?

Optimizing ITGAV detection in tissue samples requires careful consideration of fixation, antigen retrieval, and antibody incubation parameters. Based on validated protocols, the following methodological approach is recommended:

Tissue Processing and Fixation:

• Fix tissue in 10% neutral buffered formalin for 24-48 hours

• Process and embed in paraffin following standard histological procedures

• Section tissues at 4-6μm thickness onto charged slides

• Dry sections overnight at 37°C

Antigen Retrieval Optimization:

• Heat-induced epitope retrieval in citrate buffer (pH 6.0) for 20 minutes

• Allow slides to cool in buffer for 20 minutes before proceeding

• Wash in PBS (3 times, 5 minutes each)

Immunostaining Protocol:

• Block endogenous peroxidase with 3% H₂O₂ in methanol for 15 minutes

• Block non-specific binding with 5% normal serum for 1 hour

• Apply HRP-conjugated ITGAV antibody at 1:20-1:200 dilution

• Incubate in a humidified chamber at 4°C overnight

• Wash thoroughly with PBS (3 times, 5 minutes each)

• Develop with DAB substrate until optimal signal intensity (2-10 minutes)

• Counterstain with hematoxylin, dehydrate, and mount

For dual immunofluorescence applications, the HRP-conjugated ITGAV antibody can be detected using tyramide signal amplification systems to enable multiplex analysis with other markers. This approach is particularly valuable for examining ITGAV heterodimer formation with various beta partners in tissue contexts .

What controls are essential when using ITGAV Antibody, HRP conjugated to ensure experimental validity?

Rigorous controls are critical for ensuring the validity and reproducibility of experiments using ITGAV antibody. The following control strategy addresses key aspects of experimental design:

Positive Controls:

• Cell lines with known ITGAV expression (e.g., MCF-7 cells)

• Human tissue samples with established ITGAV expression patterns (placenta, vasculature)

• Recombinant ITGAV protein for Western blot standard curves

Negative Controls:

• Primary antibody omission control (all reagents except primary antibody)

• Isotype control (irrelevant HRP-conjugated IgG at matching concentration)

• Cell lines with CRISPR knockout of ITGAV

• Competitive binding with immunizing peptide (when available)

Technical Controls:

• Loading control for Western blot (β-actin, GAPDH)

• Tissue positive internal control (blood vessels typically express ITGAV)

• Serial dilution of primary antibody to confirm specificity

• Cross-reactivity assessment with related integrin alpha subunits

Validation Approaches:

• Comparison of results using alternative ITGAV antibody clones

• Correlation with mRNA expression data

• Functional validation using ITGAV inhibitors or siRNA knockdown

For flow cytometry applications specifically, unstained cells and fluorescence minus one (FMO) controls should be included to establish proper gating strategies. Validated flow cytometry protocols for ITGAV detection include using the antibody at 1:50-1:100 dilution as demonstrated with MCF-7 cells .

How can researchers differentiate between specific and non-specific binding when using ITGAV Antibody, HRP conjugated?

Distinguishing specific from non-specific signals is crucial for accurate ITGAV detection. The following methodological approach helps establish signal specificity:

Signal Verification Strategy:

• Molecular Weight Verification:

  • Confirm that the primary band detected corresponds to the calculated ITGAV molecular weight of approximately 116 kDa

  • Be aware that post-translational modifications may alter apparent molecular weight

• Peptide Competition Assay:

  • Pre-incubate the antibody with excess immunizing peptide

  • Compare signal between competed and non-competed antibody

  • Specific signals should be substantially reduced or eliminated

• Signal Correlation Analysis:

  • Compare detection patterns across multiple techniques (WB, IHC, flow cytometry)

  • Specific signals should show consistent patterns across methodologies

  • Compare results with mRNA expression data

• Genetic Validation Approaches:

  • Test antibody on samples with ITGAV knockdown/knockout

  • Specific signals should be reduced proportionally to knockdown efficiency

  • Include heterozygous samples to verify dose-dependent signal reduction

• Cross-Reactivity Assessment:

  • Test on samples expressing related integrin family members

  • Verify absence of signal in species not covered by the antibody's reactivity profile

For flow cytometry applications, non-specific binding can be minimized by:

• Using appropriate blocking sera matched to secondary antibody species

• Including dead cell discrimination dyes to eliminate autofluorescent dead cells

• Comparing with isotype control at identical concentration

What approaches should be taken when ITGAV antibody results contradict other experimental findings?

When faced with contradictory results between ITGAV antibody data and other experimental approaches, a systematic troubleshooting strategy should be employed:

Methodological Reconciliation Approach:

• Technical Validation:

  • Verify antibody quality through fresh aliquots and validation controls

  • Confirm protein loading and transfer efficiency in Western blots

  • Assess tissue fixation quality and antigen retrieval efficiency in IHC

  • Evaluate sample preparation methods for potential epitope masking

• Biological Variability Assessment:

  • Consider post-translational modifications affecting epitope recognition

  • Evaluate potential alternative splicing variants of ITGAV

  • Assess heterodimer formation status which may mask epitopes

  • Consider activation state of the integrin complex

• Contextual Reconciliation:

  • Examine differences in experimental conditions between contradictory findings

  • Consider cell type-specific regulation of ITGAV expression

  • Evaluate matrix composition differences affecting integrin conformation

  • Assess potential compensatory mechanisms in genetic models

• Cross-Validation Strategies:

  • Deploy alternative antibody clones recognizing different ITGAV epitopes

  • Utilize non-antibody detection methods (mass spectrometry, RNA-seq)

  • Combine functional assays with molecular detection

  • Implement genetic labeling approaches (CRISPR-mediated tagging)

A specific example of reconciliation involves understanding the relationship between ITGAV and its binding partners: mutations in binding partners (like ITGB5) may affect ITGAV stability or localization without altering expression levels, creating apparent contradictions between functional and expression data .

How can researchers distinguish between different ITGAV heterodimers using antibody-based approaches?

Distinguishing between different ITGAV heterodimers (αVβ3, αVβ5, αVβ6, αVβ8) is methodologically challenging but can be accomplished through:

Heterodimer Discrimination Strategy:

• Co-Immunoprecipitation Approach:

  • Immunoprecipitate with ITGAV antibody

  • Probe with specific beta subunit antibodies (ITGB3, ITGB5, ITGB6, ITGB8)

  • Quantify relative abundance of each heterodimer

• Proximity Ligation Assay (PLA):

  • Combine ITGAV antibody with specific beta subunit antibodies

  • PLA signal occurs only when proteins are within 40nm

  • Quantify distinct heterodimer populations in situ

• Function-Blocking Antibody Approach:

  • Apply selective function-blocking antibodies targeting specific heterodimers

  • Measure functional outcomes (adhesion, signaling)

  • Determine relative contribution of each heterodimer

• Heterodimer-Specific Detection:

  • Utilize conformation-specific antibodies that recognize only assembled heterodimers

  • Focus on activation state-specific epitopes that emerge upon heterodimer formation

  • Consider ligand binding domains that differ between heterodimers

• Correlation Analysis:

  • Compare ITGAV expression patterns with beta subunit distribution

  • Identify tissue/cell types with predominant expression of specific beta partners

  • Utilize the distinct ligand preference of different heterodimers

Recent research has highlighted the importance of the ITGAV-ITGB5 heterodimer in cancer cell expansion, making this specific heterodimer a particularly valuable target for discrimination studies in oncology research contexts .

How can ITGAV Antibody, HRP conjugated be leveraged to investigate the role of integrin αVβ5 in cancer progression?

Recent CRISPR screens have identified the ITGAV-ITGB5 heterodimer as the essential integrin pair for cancer cell expansion, making this a critical area for advanced investigation . The HRP-conjugated ITGAV antibody can be leveraged in several sophisticated experimental approaches:

Cancer Research Applications:

• Pharmacological Inhibitor Studies:

  • Monitor ITGAV-ITGB5 heterodimer stability following treatment with novel inhibitors like Cpd_AV2

  • Quantify heterodimer dissociation kinetics using time-course analysis

  • Correlate heterodimer stability with cancer cell apoptosis markers

• Dimerization Domain Analysis:

  • Investigate the β-propeller domain of ITGAV which contains the critical pocket for heterodimer formation

  • Track conformational changes upon ligand binding or inhibitor treatment

  • Couple with site-directed mutagenesis of key residues in the dimerization interface

• Tumor Microenvironment Interaction Studies:

  • Analyze ITGAV localization at tumor-stroma boundaries

  • Correlate ITGAV expression with extracellular matrix composition

  • Investigate ITGAV-dependent mechanosensing in tumor progression

• Signaling Pathway Integration:

  • Examine co-localization of ITGAV with downstream signaling molecules

  • Investigate phosphorylation cascades triggered by ITGAV-ITGB5 activation

  • Analyze cross-talk between integrin and growth factor receptor pathways

The CRISPR-Tiling-Instructed Computer-Aided (CRISPR-TICA) approach has identified the integral pocket within the β-propeller domain of ITGAV as critical for cancer progression, making this region a high-priority target for therapeutic development and mechanistic studies .

What methodologies can elucidate the molecular mechanisms of ITGAV heterodimer stability and dissociation?

Understanding the molecular mechanisms governing ITGAV heterodimer stability is critical for both basic science and therapeutic development. The following advanced methodological approaches can be employed:

Molecular Mechanism Analysis:

• Structural Biology Integration:

  • Combine antibody epitope mapping with structural data

  • Correlate antibody binding with heterodimer stability measurements

  • Identify conformational epitopes that appear during heterodimer dissociation

• Live Cell Imaging Approaches:

  • Utilize FRET-based sensors to monitor heterodimer proximity in real-time

  • Track heterodimer dynamics during cell migration and adhesion

  • Correlate with cellular phenotypes following perturbation

• Quantitative Binding Kinetics:

  • Measure association/dissociation rates of ITGAV with beta partners

  • Compare kinetics in the presence of various ligands or inhibitors

  • Correlate binding parameters with functional outcomes

• High-Resolution Microscopy:

  • Employ super-resolution techniques to visualize nanoscale organization of ITGAV

  • Track dynamic reorganization during cellular processes

  • Identify molecular co-clustering with signaling partners

Recent research using Cpd_AV2, which targets the β-propeller central pocket of ITGAV, demonstrated rapid uncoupling of integrin αVβ5 followed by cellular apoptosis. This provides a mechanistic model for studying heterodimer stability in various cellular contexts .

How can researchers investigate the role of ITGAV in rare genetic disorders?

ITGAV and its binding partners have been implicated in rare genetic disorders, including early-onset inflammatory bowel disease (IBD). Advanced research approaches include:

Genetic Disorder Investigation Strategy:

• Digenic Inheritance Analysis:

  • Investigate co-occurrence of ITGAV variants with partner protein mutations

  • Examine the digenic autosomal recessive inheritance pattern observed in early-onset IBD

  • Assess functional consequences of specific mutations like ITGAV (G58V)

• Genotype-Phenotype Correlation:

  • Compare clinical manifestations with specific ITGAV variant profiles

  • Develop mutation-specific antibodies for variant protein detection

  • Correlate mutation location with functional domains of ITGAV

• Conservation Analysis Approach:

  • Examine evolutionary conservation of affected residues across species

  • Predict functional impact of mutations in highly conserved regions

  • Compare with in vitro functional assays to validate predictions

• Therapeutic Response Prediction:

  • Stratify patients based on ITGAV variant status

  • Correlate variant profiles with response to integrin-targeted therapies

  • Develop personalized treatment approaches based on specific mutations

• Animal Model Development:

  • Generate knock-in models with specific ITGAV mutations

  • Compare phenotypes with human disease manifestations

  • Test potential therapeutic approaches in genetically accurate models

Exome sequencing has identified extremely rare ITGAV variants in monozygotic twins with early-onset IBD, highlighting the importance of incorporating genetic screening into research on integrin-associated disorders .

What are the recommended storage and handling conditions to maintain HRP-conjugated ITGAV antibody activity?

Proper storage and handling of HRP-conjugated ITGAV antibody is critical for maintaining its activity and ensuring reproducible results across experiments. Based on manufacturer recommendations:

Storage Conditions:

• Long-term storage: -20°C for up to one year

• Short-term storage and frequent use: 4°C for up to one month

• Avoid repeated freeze-thaw cycles that can degrade both antibody and HRP enzyme

Storage Formulation:
The standard formulation includes:

• 1mg/ml antibody concentration in PBS

• 0.02% sodium azide as preservative

• 50% glycerol for cryoprotection

• pH maintained at 7.2

Handling Practices:

• Aliquot upon first thaw to minimize freeze-thaw cycles

• Thaw at room temperature or 4°C, never at higher temperatures

• Mix gently by inversion, avoid vortexing which can denature antibodies

• Centrifuge briefly before opening to collect all liquid at the bottom

• Use sterile technique when handling to prevent microbial contamination

Quality Control Monitoring:

• Check for visible precipitates before use (indication of degradation)

• Include positive controls with known ITGAV expression in each experiment

• Monitor signal-to-noise ratio across experiments to detect potential activity loss

• Consider preparing standard curves to track sensitivity over time

Following these storage and handling guidelines will help ensure consistent antibody performance and reliable experimental results.

What is the intra-assay and inter-assay precision for ITGAV detection using ELISA with HRP-conjugated antibodies?

Precision metrics are essential for evaluating the reliability of ITGAV quantification using ELISA with HRP-conjugated antibodies. Based on validated protocols:

Intra-assay Precision (within-run precision):

• Measured by testing three samples (low, middle, and high ITGAV concentrations) 20 times on one plate

• Typical coefficient of variation (CV): <10%

• Example data showed CV values of 4.8% (low), 6.2% (middle), and 3.9% (high) concentration samples

Inter-assay Precision (between-run precision):

• Determined by testing three samples in 20 separate assays

• Typical coefficient of variation (CV): <15%

• Example data showed CV values of 8.7% (low), 10.3% (middle), and 7.6% (high) concentration samples

Precision Optimization Factors:

• Antibody quality and consistency

• Careful adherence to incubation times and temperatures

• Precise reagent preparation and pipetting

• Consistent washing technique

• Standardized plate reading parameters

Analytical Performance Characteristics:

• Detection range: 0.156-10 ng/mL (typical for ITGAV ELISA)

• Sensitivity: <0.1 ng/mL

• Recovery rate: 85-115% in spiked samples

• Linearity: r² > 0.98 across the detection range

These precision metrics ensure that ITGAV quantification is reliable and reproducible across experiments, which is critical for longitudinal studies and multi-center research collaborations.

Human Integrin Alpha V (ITGαV) ELISA Kit Performance Characteristics: