NECTIN3 Antibody

What is NECTIN3 and why is it important in research?

NECTIN3 is an 83 kDa type I transmembrane glycoprotein belonging to the immunoglobulin superfamily that functions as a calcium-independent cell adhesion molecule. It plays crucial roles in forming cell-cell junctions including adherens junctions, synaptic junctions in neurons, and Sertoli cell-spermatid junctions. NECTIN3 is significant in research due to its diverse expression pattern and involvement in multiple biological processes such as cell adhesion, neuronal development, and tissue architecture maintenance. Recent studies have linked NECTIN3 reduction to stress-induced prefrontal structural and functional changes, highlighting its potential role in stress-related neuropsychiatric conditions .

What are the structural characteristics of NECTIN3 protein that influence antibody selection?

NECTIN3 protein contains three Ig-like domains in its extracellular region: one N-terminal V-type domain and two membrane-proximal C2-type domains. The cytoplasmic region contains a Glu-Trp-Tyr-Val motif that binds afadin, an actin filament-binding protein . These structural features are important considerations for antibody selection:

When selecting antibodies, researchers should consider which domain they need to target based on their experimental goals .

What are the different isoforms of NECTIN3 and how do they affect antibody detection?

NECTIN3 exists in three isoforms: nectin-3α, -3β, and -3γ, with nectin-3α being the largest. These isoforms differ in their structure and potentially in their functions:

• Full-length isoform (nectin-3α)

• Second isoform: Has a 31 amino acid substitution for the first 54 amino acids of the signal sequence, followed by a deletion of amino acids 291-549

• Third isoform: Shows a 10 amino acid substitution for amino acids 357-549

When working with NECTIN3 antibodies, researchers must verify which isoforms the antibody can detect. Some antibodies may recognize all isoforms while others may be isoform-specific. This becomes particularly important when interpreting western blot results, where different bands may represent different isoforms rather than non-specific binding .

What are the optimal applications for different types of NECTIN3 antibodies?

Different NECTIN3 antibodies are optimized for specific applications. Based on the available data, here's a comprehensive guide:

For detecting endogenous NECTIN3 in intact cells or tissues, antibodies targeting the extracellular domain are recommended. For denatured protein detection (Western blot), antibodies recognizing linear epitopes work better .

How should researchers optimize immunohistochemistry protocols for NECTIN3 detection?

Based on published protocols, here is an optimized methodology for NECTIN3 immunohistochemistry:

• Fixation and Processing:

  • For paraffin sections: Immersion-fixed paraffin-embedded sections yield good results

  • For frozen sections: Perfusion fixation followed by cryosectioning preserves antigenicity

• Antibody Concentration and Incubation:

  • Primary antibody: 10 μg/mL for polyclonal antibodies or 1:100-1:200 dilution

  • Incubation: Overnight at 4°C for optimal binding and reduced background

• Detection System:

  • For chromogenic detection: Anti-species HRP-DAB system with hematoxylin counterstain

  • For fluorescence: Species-appropriate secondary antibodies (e.g., goat anti-rabbit-AlexaFluor-488)

• Visualization Parameters:

  • Image acquisition: 100× or 200× magnification using appropriate microscopy

  • For co-localization: Confocal microscopy at 200× (optical section thickness, 0.362 μm) or 600× (optical section thickness, 0.196 μm) using sequential scanning mode

For optimal results, sections should be randomly numbered for unbiased analysis, and the brightness and contrast of images should be standardized using appropriate software (e.g., FV10-ASW 1.7) .

What are the critical considerations for Western blot analysis of NECTIN3?

Western blot analysis of NECTIN3 requires attention to several important factors:

• Sample Preparation:

  • Membrane fraction preparation is often necessary as NECTIN3 is a membrane protein

  • Complete denaturation is crucial for accurate molecular weight determination

• Expected Molecular Weight:

  • Full-length NECTIN3: ~83 kDa

  • Lower molecular weight bands may represent isoforms or proteolytic fragments

• Blocking and Antibody Dilutions:

  • Optimal blocking: 5% non-fat milk or BSA in TBST

  • Primary antibody dilutions: 1:200 to 1:1000 depending on the specific antibody

• Validation Controls:

  • Positive controls: Testis membranes, brain lysates, and U-87 MG glioblastoma cell lines have shown reliable NECTIN3 expression

  • Negative control: Preincubation with specific blocking peptide to demonstrate specificity

  • Additional control: Samples from NECTIN3 knockout models or knockdown cells

• Tissue-Specific Considerations:

  • Brain tissues may show different banding patterns compared to testis or other tissues due to differential post-translational modifications and isoform expression

How can researchers effectively utilize NECTIN3 antibodies for studying stress-induced neurological changes?

Recent research has identified NECTIN3 as a potential mediator of adolescent chronic stress effects on prefrontal structure and function. Researchers interested in this area should consider:

• Experimental Design:

  • Use age-appropriate models (adolescent animals for developmental studies)

  • Include adequate sample sizes (n=14-16 per group based on previous protocols)

  • Plan for both behavioral and molecular analyses from the same cohort

• Knockdown Validation:

  • AAV-shNectin3 viral vectors can be used to suppress NECTIN3 levels (target sequence: 5′-TGTGTCCTGGAGGCGGCAAAGCACAACTT-3′)

  • Inject virus (3.9 × 10^12 viral genomes/mL) into specific brain regions using stereotactic surgery

  • Coordinates for mouse mPFC: anterior +1.8 mm, lateral ±0.4 mm, ventral −1.8 mm relative to bregma

  • Verify knockdown efficiency through immunoreactivity measurements and Western blot

• Analysis Methods:

  • Quantify immunoreactivity using ImageJ software

  • Calculate relative protein levels as differences in optical density between the region of interest and a background region (e.g., corpus callosum)

  • Normalize results by taking the mean value of the control group as 100%

This approach can help elucidate NECTIN3's role in stress-related neuropsychiatric conditions and identify potential therapeutic targets.

What strategies can resolve antibody cross-reactivity issues with other nectin family members?

Cross-reactivity with other nectin family members can complicate NECTIN3 antibody experiments. Here are methodological approaches to address this issue:

• Antibody Selection and Validation:

  • Choose antibodies with documented low cross-reactivity (<5%) with other nectin family members

  • Perform direct ELISA against recombinant proteins of all nectin family members to quantify cross-reactivity

  • Use nectin-3 knockout samples as definitive negative controls

• Epitope Targeting:

  • Target regions with low sequence homology between nectin family members

  • Avoid antibodies targeting the highly conserved Ig-like domains if specificity is crucial

• Complementary Approaches:

  • Use multiple antibodies targeting different epitopes to confirm results

  • Implement genetic approaches (siRNA, CRISPR) alongside antibody-based detection

  • Perform immunodepletion studies to verify specificity

• Data Analysis Considerations:

  • Always include positive and negative controls in experimental design

  • Use quantitative methods for signal measurement rather than qualitative assessment

  • Apply appropriate statistical analyses to determine significance of findings

How can researchers investigate NECTIN3 interactions with binding partners using antibody-based techniques?

NECTIN3 forms heterophilic trans-interactions with multiple binding partners, including nectin-1, nectin-2, and PVR. To study these interactions:

• Co-immunoprecipitation (Co-IP):

  • Use antibodies targeting distinct epitopes from the interaction domains

  • For afadin interactions, use antibodies targeting the N-terminal region of NECTIN3

  • Apply gentle lysis conditions to preserve protein-protein interactions

  • Verify results with reverse Co-IP (precipitate with partner protein antibody)

• Proximity Ligation Assay (PLA):

  • Enables visualization of protein interactions in situ with subcellular resolution

  • Requires antibodies from different host species for each interaction partner

  • Quantify interaction signals as discrete fluorescent dots using image analysis software

• FRET/BRET Approaches:

  • Label NECTIN3 and binding partners with fluorescent/bioluminescent tags

  • Use antibodies to verify correct expression and localization

  • Measure energy transfer to determine molecular proximity in living cells

• Functional Blocking Studies:

  • Certain antibodies (e.g., 103-A1) can disrupt NECTIN3 interactions when injected into tissues

  • This approach can reveal the functional significance of specific interactions

  • Monitor phenotypic changes such as disruption of actin filaments or exfoliation of spermatids

How should researchers interpret variations in NECTIN3 expression across different tissues and experimental conditions?

NECTIN3 shows diverse expression patterns across tissues. When interpreting experimental results:

• Tissue-Specific Expression Profiles:

  • High expression: Testis, placental tissues, junctions between small intestinal columnar epithelial cells

  • Neuronal expression: Synaptic junctions, spinal cord motor neurons, axons, prefrontal cortex

  • Special structures: Pigmented and nonpigmented epithelium in the ciliary body

• Cellular Localization Patterns:

  • Primarily anchors to postsynaptic membrane at puncta adherentia junctions (PAJs)

  • Forms heterophilic adhesions with presynaptic nectin-1

  • Connects to actin cytoskeleton via afadin

• Expression Changes in Pathological Conditions:

  • Stress-induced reduction correlates with prefrontal structural changes

  • NECTIN3 knockdown can mimic stress effects on behavior and morphology

  • Changes should be quantified relative to appropriate controls and normalized to housekeeping proteins

• Experimental Variables to Consider:

  • Age-dependent expression patterns

  • Cell type-specific expression within heterogeneous tissues

  • Post-translational modifications affecting antibody recognition

What are the key considerations when selecting positive and negative controls for NECTIN3 antibody validation?

Proper controls are essential for antibody validation. For NECTIN3 antibodies:

For knockout validation, AAV-shNectin3 (target sequence: 5′-TGTGTCCTGGAGGCGGCAAAGCACAACTT-3′) can be used to generate knockdown models, with a verified infection rate of approximately 62% in mPFC tissue .

How can researchers integrate NECTIN3 antibody data with other experimental approaches to gain comprehensive insights?

To maximize research impact, NECTIN3 antibody-based studies should be integrated with complementary approaches:

• Multidimensional Analysis Framework:

  • Molecular level: Antibody detection + transcriptomics (RNA-seq/qPCR)

  • Cellular level: Immunostaining + live cell imaging + electrophysiology

  • Systems level: Behavioral testing + connectomics

• Integration Strategies:

  • Correlate protein expression levels with functional outcomes

  • Use genetic manipulation (AAV-shRNA, CRISPR) to validate antibody findings

  • Combine in vitro and in vivo approaches to establish biological relevance

• Quantitative Analysis Methods:

  • Perform image quantification using standardized software (ImageJ/FIJI)

  • Calculate relative protein levels as differences in optical density

  • Normalize results against appropriate reference standards

  • Apply statistical analysis to determine significance (t-tests, ANOVA as appropriate)

• Mechanistic Investigations:

  • Study interactions with other molecules (e.g., afadin, nectins, PVR)

  • Investigate signaling pathways (CDC42 and RAC small G proteins, SRC, RAP1)

  • Examine effects on cellular processes (cell adhesion, migration, morphology)

By integrating these approaches, researchers can develop a comprehensive understanding of NECTIN3's roles in both normal physiology and pathological conditions.

How can NECTIN3 antibodies be utilized to investigate neurodevelopmental processes and stress-related pathologies?

NECTIN3 has emerged as a potential mediator of stress effects on prefrontal structural and functional development. Researchers can employ NECTIN3 antibodies to investigate:

• Developmental Trajectory Analysis:

  • Map NECTIN3 expression changes during critical periods of brain development

  • Correlate expression with synaptogenesis and circuit formation

  • Compare normal development with stress-exposed models

• Stress-Induced Molecular Changes:

  • Quantify NECTIN3 reduction following different stress paradigms

  • Analyze co-localization with stress-related markers (CRH, glucocorticoid receptors)

  • Examine regional specificity of stress effects across brain regions

• Cell-Type Specific Analysis:

  • Use dual immunolabeling to determine expression in excitatory versus inhibitory neurons

  • Examine expression in glia versus neurons

  • Investigate subcellular localization changes following stress exposure

• Translational Relevance:

  • Correlate NECTIN3 changes with behavioral outcomes (social approach, memory tests)

  • Test interventions aimed at normalizing NECTIN3 levels

  • Compare findings across species to establish translational validity

This research direction holds promise for understanding mechanisms underlying stress-related neuropsychiatric conditions and identifying novel therapeutic targets.

What methodological approaches can resolve discrepancies in NECTIN3 antibody results between different studies?

When researchers encounter conflicting results using NECTIN3 antibodies across studies, several methodological approaches can help resolve these discrepancies:

• Antibody Characterization Matrix:

  • Create a comprehensive table comparing antibodies across studies

  • Document epitope regions, host species, clonality, and validation methods

  • Assess batch-to-batch variability through lot number tracking

• Standardized Protocol Development:

  • Implement consistent fixation and antigen retrieval methods

  • Standardize antibody concentrations and incubation conditions

  • Use identical detection systems and quantification methods

• Multi-antibody Validation Approach:

  • Test multiple antibodies targeting different epitopes of NECTIN3

  • Compare monoclonal versus polyclonal antibodies for the same application

  • Validate with genetic approaches (knockdown, knockout)

• Systematic Analysis of Variables:

  • Tissue preparation methods (fresh vs. fixed, paraffin vs. frozen)

  • Buffer compositions and pH conditions

  • Blocking reagents and detection systems

  • Species and strain differences in the research models used

By implementing these approaches, researchers can identify sources of variability and establish more reproducible protocols for NECTIN3 detection.

How can researchers leverage recombinant NECTIN3 proteins in antibody validation and functional studies?

Recombinant NECTIN3 proteins are valuable tools for antibody validation and functional studies:

• Antibody Validation Applications:

  • Use as positive controls in Western blots and ELISAs

  • Create standard curves for quantitative analysis

  • Pre-absorb antibodies to test specificity

  • Generate blocking peptides for specificity controls

• Functional Binding Assays:

  • Measure binding affinities between NECTIN3 and interaction partners

  • Available recombinant protein: Extracellular domain (Met1-Asp400) with polyhistidine tag

  • Demonstrated binding to biotinylated Nectin-1 (linear range: 6.4-800 ng/ml)

  • Binds Nectin-1/Fc chimera with linear range of 0.156-5 ng/ml

• Advanced Applications:

  • Surface Plasmon Resonance (SPR) to measure real-time binding kinetics

  • ELISA-based interaction screening to identify novel binding partners

  • Competition assays to map binding interfaces

  • Structure-function studies using domain-specific constructs

• Production Considerations:

  • Expression systems: HEK-293 cells provide proper folding and post-translational modifications

  • Quality control: >98% purity by SDS-PAGE

  • Endotoxin testing: <1.0 EU per μg by LAL method

  • Storage: Aliquot to avoid freeze-thaw cycles