NPY2 Antibody

What is NPY2R and why is it significant in neuroscience research?

NPY2R (Neuropeptide Y Receptor Y2) is a member of the G-protein coupled receptor 1 family that functions as a receptor for neuropeptide Y and peptide YY. It is predominantly expressed in the brain and has been implicated in various neurological conditions, including cocaine dependence and nephronophthisis 4 . The significance of NPY2R in neuroscience research stems from its role in multiple physiological processes and its potential as a therapeutic target. Researchers investigating NPY2R typically employ immunohistochemistry, western blotting, and immunocytochemistry to detect and characterize this receptor in experimental models.

How are NPY2R antibodies generated for research applications?

NPY2R antibodies are generated through two primary approaches: immunization with purified, enriched receptors or with receptor fragments . For monoclonal antibodies like the L119/106 clone, researchers typically produce hybridoma lines using specific antigens, such as fusion proteins containing amino acids 1-51 (extracellular N-terminus) and 330-381 (cytoplasmic C-terminus) of human NPY2R, recombinantly produced in E. coli . The hybridoma cells are cultured in vitro in bioreactors, followed by Protein A affinity chromatography for purification. This process yields highly specific antibodies (>90% specific) with recognition capability across human, mouse, and rat NPY2R proteins . These production methods are critical for ensuring antibody specificity and reproducibility in experimental applications.

What species reactivity and cross-reactivity should be considered when selecting NPY2R antibodies?

When selecting NPY2R antibodies, researchers should carefully consider both the target species of their study and potential cross-reactivity issues. The commercially available Anti-NPY2R monoclonal antibody (L119/106) has confirmed reactivity with human, mouse, and rat NPY2R . This cross-species reactivity makes it valuable for comparative studies across these commonly used research models.

Key: (-) no fluorescence; (+) weak fluorescence; (++) moderate fluorescence; (+++) strong fluorescence

What are the optimal protocols for immunohistochemical detection of NPY2R in tissue samples?

For optimal immunohistochemical detection of NPY2R in tissue samples, researchers should follow a validated protocol that maximizes signal specificity while minimizing background. Based on published research applications, a recommended protocol includes:

• Tissue fixation: Use 4% paraformaldehyde in phosphate-buffered saline (PBS) for 24 hours at 4°C.

• Tissue processing: Dehydrate, clear, and embed in paraffin; alternatively, cryopreservation can be employed for frozen sections.

• Sectioning: Cut 5-7 μm sections and mount on positively charged slides.

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

• Blocking: Incubate sections with 5-10% normal serum (matching the species of secondary antibody) with 0.1% Triton X-100 in PBS for 1 hour at room temperature.

• Primary antibody incubation: Apply anti-NPY2R antibody (L119/106) at an optimized dilution (typically starting at 1:100-1:500) overnight at 4°C .

• Secondary antibody: Use appropriate species-specific biotinylated or fluorophore-conjugated secondary antibody.

• Signal detection: For chromogenic detection, employ streptavidin-HRP followed by DAB; for fluorescence, directly visualize using appropriate filters.

• Counterstaining: Use hematoxylin for brightfield or DAPI for fluorescence.

This protocol has been successfully applied in studies examining NPY2R expression in newly formed blood vessels, demonstrating that Y2 receptor protein is present in virtually all newly formed blood vessels induced by NPY, FGF-2, and VEGF .

How can researchers differentiate between NPY receptor subtypes using antibody-based approaches?

Differentiating between NPY receptor subtypes requires careful selection of antibodies with proven subtype specificity. Research has demonstrated that a combination of antibodies targeting different extracellular loops can be used to distinguish between Y1-, Y2-, and Y5-receptor subtypes .

The most effective approach involves:

• Selecting antibodies with validated subtype selectivity: For example, sera against the E2 loop of the Y1-receptor and against the E2 loop of the Y2-receptor have demonstrated subtype selectivity .

• Employing multiplexed immunofluorescence: Using differentially labeled secondary antibodies to visualize multiple receptor subtypes simultaneously in the same tissue section.

• Including appropriate controls: Testing antibodies on cells or tissues with known expression profiles of different NPY receptor subtypes. The cell lines SK-N-MC (Y1-receptor), SMS-KAN (Y2-receptor), and BHK (Y5-receptor) can serve as valuable controls .

• Complementary validation: Combining antibody-based detection with functional assays or RNA expression analysis to confirm subtype-specific expression.

• Western blot analysis: Confirming receptor identity based on molecular weight (approximately 45 kDa for NPY2R) .

This multi-faceted approach enables researchers to reliably distinguish between NPY receptor subtypes, which is essential for understanding their distinct physiological roles and potential as therapeutic targets.

What controls should be included when using NPY2R antibodies in experimental designs?

Rigorous experimental design for NPY2R antibody applications requires comprehensive controls to ensure result validity and interpretability:

• Positive controls:

  • Tissues or cell lines with confirmed NPY2R expression (e.g., SMS-KAN cells for Y2-receptor)

  • Rat whole brain lysate, which has been validated for western blotting with anti-NPY2R antibody (L119/106)

  • Newly formed blood vessels, which demonstrate robust Y2 receptor expression

• Negative controls:

  • Primary antibody omission to assess non-specific binding of secondary antibodies

  • Y2 receptor-null (Y2-/-) mice tissues, which provide an excellent specificity control

  • Cell lines lacking Y2-receptor expression (e.g., SK-N-MC, which expresses Y1 but not Y2 receptors)

• Specificity controls:

  • Preabsorption of antibody with immunizing peptide to confirm epitope-specific binding

  • Sequential dilution series to evaluate optimal antibody concentration

  • Cross-reactivity testing with related receptor subtypes (Y1, Y4, Y5) to ensure specificity

• Technical controls:

  • Isotype control antibodies (matching IgG2a for L119/106) to assess Fc-mediated binding

  • Internal loading controls for western blot applications

  • Multiple reference genes for complementary qPCR validation of receptor expression

Incorporating these controls ensures that observed signals represent genuine NPY2R expression rather than technical artifacts or cross-reactivity with other receptor subtypes.

How does NPY2R expression change during angiogenesis and wound healing processes?

NPY2R expression undergoes significant changes during angiogenesis and wound healing processes, playing a crucial role in vascular development and tissue repair. Advanced research has revealed:

• Vascular expression patterns: Y2 receptor protein is present in virtually all newly formed blood vessels induced not only by NPY but also by other angiogenic factors including FGF-2 and VEGF . This widespread expression suggests a general role for NPY2R in regulating angiogenesis across different physiological and pathological contexts.

• Temporal dynamics: During wound healing, NPY2R expression increases in the proliferative phase, coinciding with active angiogenesis. This temporal correlation supports its functional relevance in the repair process.

• Cellular specificity: While Y2 receptor is widely distributed on newly formed blood vessels, the other NPY receptor subtypes (Y1, Y4, Y5) are not detected in corneal capillaries induced by NPY, FGF-2, and VEGF . This selective expression pattern underscores the specific role of NPY2R in vascular development.

• Functional significance: Y2 receptor-deficient (Y2-/-) mice exhibit:

  • Complete absence of NPY-induced corneal blood vessel growth

  • Significantly delayed skin wound healing (p < 0.001)

  • Reduced neovascularization in wound tissues

  • Inability to respond to exogenous NPY application, while maintaining normal response to FGF-2

These findings collectively demonstrate that NPY2R expression is dynamically regulated during angiogenesis and wound healing, with its presence being essential for NPY-mediated vascular formation and tissue repair.

What experimental approaches can resolve contradictory findings about NPY2R localization?

Resolving contradictory findings about NPY2R localization requires a multi-faceted experimental approach that combines complementary techniques and rigorous controls:

• Multi-epitope antibody validation:

  • Employ antibodies targeting different epitopes of NPY2R (e.g., N-terminal, C-terminal, and extracellular loops)

  • Compare localization patterns to identify consensus sites of expression

  • The fusion protein approach used for the L119/106 antibody, which incorporates both N-terminal (amino acids 1-51) and C-terminal domains (amino acids 330-381), provides enhanced epitope recognition

• Genetic models and knockdown approaches:

  • Utilize Y2 receptor-null mice as negative controls for antibody specificity

  • Employ conditional knockout models to examine tissue-specific localization patterns

  • Implement siRNA or shRNA knockdown in cell culture systems to validate antibody specificity

• Orthogonal detection methods:

  • Complement immunohistochemistry with in situ hybridization to detect NPY2R mRNA

  • Employ receptor autoradiography with NPY2R-selective radioligands

  • Utilize fluorescent NPY analogs with subtype selectivity for Y2 receptor

• Subcellular fractionation and biochemical verification:

  • Perform western blotting on membrane, cytosolic, and nuclear fractions

  • Use surface biotinylation to specifically identify plasma membrane-associated receptors

  • Employ proteomic approaches to identify receptor-associated protein complexes

• Live-cell imaging techniques:

  • Generate fluorescently tagged NPY2R constructs for real-time visualization

  • Employ FRET or BRET approaches to examine receptor dimerization and trafficking

By systematically integrating these approaches, researchers can distinguish genuine localization patterns from artifacts and reconcile apparently contradictory findings about NPY2R distribution in different tissues and experimental systems.

How can researchers distinguish between direct and indirect effects when studying NPY2R signaling pathways?

Distinguishing between direct and indirect effects in NPY2R signaling pathways requires sophisticated experimental design and careful interpretation:

• Receptor-specific pharmacological tools:

  • Utilize Y2 receptor-specific agonists and antagonists

  • The NPY analogue [Leu31Pro34]NPY, which lacks high affinity for the NPY Y2 receptor but stimulates Y1 and Y5 receptors, can help differentiate receptor subtype-specific effects

  • Compare responses to full-length NPY versus C-terminal fragments with Y2 receptor selectivity

• Genetic approaches:

  • Compare signaling responses in wild-type versus Y2-/- models

  • In the corneal angiogenesis model, NPY completely failed to induce blood vessel growth in Y2 receptor knockout mice, while FGF-2 and VEGF retained their angiogenic activity, demonstrating the specific requirement for Y2 receptor in NPY-induced angiogenesis

  • Employ receptor chimeras to identify domains responsible for specific signaling cascades

• Temporal resolution of signaling events:

  • Perform time-course analyses to establish the sequence of signaling events

  • Use rapid kinetic approaches to identify primary versus secondary signaling responses

  • Implement phosphoproteomic approaches to map early signaling events

• Cell-autonomous versus non-cell-autonomous effects:

  • Conduct co-culture experiments with receptor-expressing and non-expressing cells

  • Employ tissue-specific conditional knockout models

  • Use cell type-specific expression systems in vivo

• Direct signaling pathway interrogation:

  • Employ selective inhibitors of downstream signaling molecules

  • Use CRISPR/Cas9-mediated gene editing to modify specific signaling components

  • Implement biosensor approaches to monitor real-time signaling dynamics

These methodological approaches collectively provide a framework for distinguishing direct NPY2R-mediated signaling events from indirect effects mediated by secondary messengers or intercellular communication.

What are the common pitfalls in NPY2R antibody-based experiments and how can they be avoided?

Researchers encounter several common pitfalls when working with NPY2R antibodies. Here are the most significant challenges and strategies to overcome them:

• Non-specific binding and background issues:

  • Pitfall: High background signal obscuring specific NPY2R detection

  • Solution: Optimize blocking conditions (use 5-10% serum matching secondary antibody species, plus 0.1-0.3% Triton X-100); include 0.1-0.2% BSA in antibody diluent; thoroughly wash samples between steps

• Antibody cross-reactivity:

  • Pitfall: Some antibodies against Y5 receptor (Y5 E2/2 and Y5 E3) show strong cross-reactivity with Y2 receptor

  • Solution: Validate antibody specificity using Y2 receptor-null tissues; perform parallel experiments with multiple antibodies targeting different epitopes; include appropriate positive and negative controls

• Fixation-related epitope masking:

  • Pitfall: Overfixation can mask epitopes and reduce antibody binding

  • Solution: Optimize fixation protocols; perform antigen retrieval; test multiple fixatives if possible; consider using fresh frozen tissues for sensitive epitopes

• Batch-to-batch variability:

  • Pitfall: Variable results between antibody lots

  • Solution: Document lot numbers; validate each new lot against previous standards; purchase sufficient quantity of a single lot for extended studies

• Suboptimal storage and handling:

  • Pitfall: Antibody degradation leading to reduced sensitivity

  • Solution: Store antibodies according to manufacturer recommendations (typically ≤ -20°C for long term storage or 2-8°C for short term); avoid repeated freeze-thaw cycles; aliquot stock solutions; centrifuge vials before opening

• Inadequate signal amplification:

  • Pitfall: Weak detection of low-abundance receptors

  • Solution: Employ signal amplification systems (e.g., tyramide signal amplification); optimize antibody concentration and incubation time; use high-sensitivity detection systems

• Validation gaps:

  • Pitfall: Insufficient validation leading to unreliable results

  • Solution: Include key controls (Y2-/- tissue, peptide competition, isotype controls); complement antibody detection with orthogonal methods (RT-PCR, functional assays)

By anticipating these common pitfalls and implementing the suggested solutions, researchers can significantly improve the reliability and interpretability of their NPY2R antibody-based experiments.

How should researchers interpret discrepancies between protein and mRNA expression of NPY2R?

Interpreting discrepancies between NPY2R protein (detected by antibodies) and mRNA expression requires careful consideration of multiple biological and technical factors:

• Post-transcriptional regulation mechanisms:

  • microRNA regulation: Investigate whether NPY2R transcripts are targeted by miRNAs that inhibit translation without affecting mRNA levels

  • RNA stability factors: Examine whether RNA-binding proteins affect transcript stability versus translation efficiency

  • Alternative splicing: Assess whether splice variants affect antibody recognition but not mRNA detection primers

• Post-translational regulation:

  • Receptor internalization and recycling: Determine whether NPY2R undergoes regulated trafficking affecting antibody detection

  • Protein stability differences: Investigate whether protein degradation rates differ between experimental conditions

  • Post-translational modifications: Examine whether modifications alter antibody epitope recognition

• Methodological considerations:

  • Detection sensitivity differences: Compare detection limits of antibody-based methods versus mRNA detection

  • Sampling differences: Ensure that protein and RNA samples represent equivalent biological material

  • Temporal dynamics: Consider time lags between transcription and translation; perform time-course studies

• Analytical approach:

  • Quantitative comparison: Perform careful quantitative analysis rather than qualitative assessment

  • Single-cell resolution: Consider single-cell approaches to address cellular heterogeneity

  • Multiple detection methods: Employ different antibodies and RNA detection methods to confirm discrepancies

• Reconciliation strategies:

  • Functional validation: Use receptor activity assays to determine whether protein or mRNA better predicts function

  • Protein synthesis inhibitors: Use cycloheximide to block translation and assess receptor turnover

  • Transcription inhibitors: Use actinomycin D to block transcription and assess mRNA stability

By systematically addressing these factors, researchers can transform apparent discrepancies between protein and mRNA expression into valuable insights about NPY2R regulation in physiological and pathological contexts.

What are the best approaches for quantifying NPY2R expression levels in different experimental models?

Quantifying NPY2R expression levels requires selecting appropriate methodologies based on experimental questions and available samples. The following approaches provide complementary information about receptor abundance:

• Western blot quantification:

  • Advantages: Provides information about receptor molecular weight (45 kDa for NPY2R ) and potential post-translational modifications

  • Methodology: Use purified recombinant NPY2R protein to generate standard curves; normalize to appropriate loading controls; employ near-infrared fluorescent detection for wider linear range

  • Considerations: Requires proper membrane protein extraction; may detect denatured epitopes not accessible in fixed tissues

• Quantitative immunohistochemistry:

  • Advantages: Preserves spatial information; allows cell type-specific quantification

  • Methodology: Use consistent image acquisition parameters; employ automated image analysis software; include calibration standards in each experiment

  • Considerations: Influenced by tissue processing variables; requires careful background correction

• Flow cytometry:

  • Advantages: Provides single-cell resolution; allows multi-parameter analysis

  • Methodology: Use indirect immunofluorescence with validated NPY2R antibodies; include appropriate isotype controls

  • Considerations: Requires efficient cell dissociation protocols; membrane integrity is critical

• ELISA-based approaches:

  • Advantages: Highly quantitative; amenable to high-throughput screening

  • Methodology: Develop sandwich ELISA using validated antibody pairs; include recombinant standards

  • Considerations: May require development of custom assays; limited spatial information

• Quantitative PCR (qPCR):

  • Advantages: Highly sensitive; can detect low-abundance transcripts

  • Methodology: Design intron-spanning primers; validate PCR efficiency; use multiple reference genes

  • Considerations: Measures mRNA rather than protein; requires validation at protein level

• Receptor binding assays:

  • Advantages: Measures functional receptors; can determine receptor density

  • Methodology: Use Y2-selective radioligands or fluorescent ligands; perform saturation binding studies

  • Considerations: Requires live cells or fresh membrane preparations; influenced by receptor conformational state

For most comprehensive assessment, researchers should employ multiple complementary approaches, with selection guided by:

• Required sensitivity (western blot detection limit approximately 10-20 ng protein)

• Need for spatial information (preserved in immunohistochemistry)

• Single-cell versus population measurements

• Available sample quantities

• Need to distinguish receptor subtypes (antibody specificity critical)

How might single-cell analysis technologies advance our understanding of NPY2R expression and function?

Single-cell analysis technologies offer unprecedented opportunities to resolve heterogeneity in NPY2R expression and function across diverse cell populations. Future research in this direction holds significant promise:

• Single-cell RNA sequencing applications:

  • Mapping cell type-specific NPY2R expression across tissues and developmental stages

  • Identifying previously unrecognized NPY2R-expressing cell populations

  • Characterizing co-expression patterns with other receptors and signaling molecules

  • Tracking transcriptional changes in NPY2R-expressing cells during pathological processes

• Single-cell proteomics:

  • Quantifying NPY2R protein abundance at single-cell resolution

  • Identifying cell type-specific post-translational modifications

  • Mapping receptor interactions with signaling partners in different cell types

• Spatial transcriptomics and proteomics:

  • Preserving spatial information while achieving single-cell resolution

  • Mapping NPY2R expression in relation to tissue microenvironment

  • Investigating regional heterogeneity in complex tissues like brain and vasculature, where Y2 receptor expression is particularly relevant

• Functional single-cell analysis:

  • Correlating NPY2R expression levels with functional responses in individual cells

  • Employing calcium imaging or other functional readouts with simultaneous receptor quantification

  • Using optogenetic approaches to manipulate NPY2R signaling in specific cells

• Computational integration:

  • Developing algorithms to integrate single-cell NPY2R data across modalities

  • Creating predictive models of receptor regulation and function

  • Building cell type-specific signaling networks centered on NPY2R

These advanced single-cell approaches will likely resolve current contradictions in NPY2R research and uncover novel biological insights, particularly in understanding the receptor's role in angiogenesis and wound healing, where cellular heterogeneity is pronounced .

What emerging technologies might improve NPY2R antibody specificity and sensitivity?

Several emerging technologies hold promise for improving NPY2R antibody specificity and sensitivity, addressing current limitations in receptor detection and characterization:

• Recombinant antibody engineering:

  • Developing single-chain variable fragments (scFvs) with enhanced epitope specificity

  • Engineering bispecific antibodies targeting multiple NPY2R epitopes simultaneously

  • Employing affinity maturation techniques to enhance binding strength while maintaining specificity

• Nanobody technology:

  • Developing camelid-derived single-domain antibodies (nanobodies) against NPY2R

  • Leveraging their small size (15 kDa) for improved tissue penetration

  • Utilizing their ability to recognize hidden or conformational epitopes

• Aptamer-based detection:

  • Developing DNA or RNA aptamers with high affinity and specificity for NPY2R

  • Combining aptamers with antibodies for dual-recognition approaches

  • Employing SELEX (Systematic Evolution of Ligands by Exponential Enrichment) to generate Y2 receptor-specific aptamers

• Proximity labeling approaches:

  • Using enzyme-mediated proximity labeling (BioID, APEX) to identify proteins in close proximity to NPY2R

  • Developing antibodies against validated proximity partners as orthogonal detection methods

  • Implementing multiplexed labeling strategies for complex interaction networks

• Advanced microscopy techniques:

  • Super-resolution microscopy for nanoscale localization of NPY2R

  • Expansion microscopy for physical magnification of structures

  • Correlative light and electron microscopy for ultrastructural contextualization

• AI-assisted epitope selection:

  • Employing machine learning algorithms to predict optimal antigenic determinants

  • Developing antibodies against computationally validated epitopes

  • Using in silico approaches to predict and minimize cross-reactivity

These technological advances will facilitate more precise detection of NPY2R in complex tissues, enabling researchers to address sophisticated questions about receptor localization, trafficking, and functional interactions in both physiological and pathological contexts.

How can systems biology approaches integrate NPY2R antibody data with other -omics datasets?

Systems biology approaches offer powerful frameworks for integrating NPY2R antibody data with other -omics datasets, providing comprehensive insights into receptor function within broader biological networks:

• Multi-omics data integration:

  • Combining antibody-based protein detection with transcriptomics, proteomics, metabolomics, and epigenomics

  • Creating multi-dimensional regulatory networks centered on NPY2R

  • Identifying unexpected correlations between NPY2R expression and other biological variables

• Network analysis approaches:

  • Constructing protein-protein interaction networks around NPY2R

  • Identifying signaling hubs and feedback loops within NPY2R signaling pathways

  • Modeling information flow through NPY2R-centered networks

• Predictive modeling:

  • Developing mathematical models of NPY2R-mediated cellular responses

  • Creating predictive algorithms for receptor behavior under various perturbations

  • Generating testable hypotheses about emergent properties of NPY2R signaling

• Comparative systems analysis:

  • Examining NPY2R networks across species, tissues, and disease states

  • Identifying conserved and divergent aspects of receptor function

  • Leveraging cross-species differences to understand fundamental receptor biology

• Temporal dynamics analysis:

  • Integrating time-course data to understand NPY2R regulation dynamics

  • Modeling receptor adaptation, sensitization, and desensitization

  • Creating dynamic visualizations of NPY2R signaling events

• Translational applications:

  • Identifying biomarkers associated with altered NPY2R function

  • Discovering potential therapeutic targets within NPY2R networks

  • Predicting off-target effects of drugs targeting NPY2R pathways

This integrated systems approach could be particularly valuable for understanding NPY2R's role in complex processes such as angiogenesis and wound healing, where the receptor appears to play a critical role . By placing NPY2R antibody data within the context of broader biological networks, researchers can develop more comprehensive models of receptor function that span from molecular interactions to physiological outcomes.