NPY4 Antibody

What is NPY4R and why is it significant for research?

NPY4R (neuropeptide Y receptor type 4) is a G protein-coupled receptor with a seven-transmembrane structure. In humans, the canonical protein has 375 amino acid residues and a mass of 42.2 kDa . It is primarily localized in the cell membrane and functions as a receptor for PPY (pancreatic polypeptide), NPY (neuropeptide Y), and PYY (peptide YY) . The receptor is notably expressed in soft tissue, small intestine, skin, rectum, and colon .

Research significance lies in its involvement in appetite regulation and stress response pathways . The receptor is negatively coupled to cAMP, making it an important component in cellular signaling mechanisms . Understanding NPY4R function has implications for disorders related to appetite dysregulation and stress-related conditions, making it a valuable target for neuroscience and endocrinology research.

What applications are NPY4R antibodies commonly used for?

NPY4R antibodies have been validated for multiple research applications:

• Western Blot (WB): For detecting NPY4R protein expression levels in cell and tissue lysates

• Immunohistochemistry (IHC): For visualizing NPY4R distribution in tissue sections

• Enzyme-Linked Immunosorbent Assay (ELISA): For quantitative detection of NPY4R

• Immunocytochemistry (ICC): For cellular localization studies

• Immunofluorescence (IF): For high-resolution imaging of NPY4R distribution

For optimal results, antibody selection should align with the specific application. For instance, polyclonal antibodies like CAB8143 have been validated for Western blot applications with a recommended dilution range of 1:500 - 1:2000 .

What sample types are compatible with NPY4R antibodies?

NPY4R antibodies have demonstrated reactivity with various sample types:

• Cell lines: MCF7, SH-SY5Y, SW480, and HT-29 have been validated as positive samples for NPY4R detection

• Primary cultures: Rat dorsal root ganglion (DRG) neurons have been successfully used for immunocytochemical staining with NPY4R antibodies

• Tissue samples: Particularly from regions with known NPY4R expression such as soft tissue, small intestine, skin, rectum, and colon

When working with new sample types, validation experiments should be conducted to confirm antibody specificity before proceeding with full experimental analysis.

How should NPY4R antibodies be stored and handled for optimal results?

For lyophilized NPY4R antibodies:

• Upon arrival, store at -20°C

• Reconstitute with double distilled water (50 μL or 0.2 mL depending on sample size)

• The reconstituted solution can be stored at 4°C for up to 1 week

• For longer storage, prepare small aliquots and store at -20°C

• Avoid multiple freeze-thaw cycles to preserve antibody activity

• Centrifuge all antibody preparations before use (10000 x g for 5 min)

Following these storage and handling guidelines ensures maintained antibody sensitivity and specificity throughout your research project.

How can researchers distinguish between different NPY receptor subtypes?

Distinguishing between NPY receptor subtypes (Y1, Y2, Y4, Y5) requires carefully selected tools and methods:

• Subtype-selective antibodies: Certain antibodies show strong selectivity for specific receptor subtypes. For example:

  • Antibody Y1 E2/2 shows selective binding for the Y1-receptor

  • Antibody Y2 E2/1 demonstrates selective binding to the Y2-receptor

  • Antibodies Y5 E2/2 and Y5 E3 bind strongly to both Y2 and Y5 receptors

• Comparative immunofluorescence: The following table summarizes receptor selectivity based on immunofluorescence assays:

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

By employing a combination of these antibodies, researchers can effectively distinguish between receptor subtypes in experimental settings.

What approaches can be used to study NPY4R ligand binding characteristics?

Several methodological approaches can be employed to study NPY4R ligand binding:

• Ala-substituted NPY analogues: These can provide insights into binding affinity and receptor selectivity. For example:

  • Substitution of proline residues at positions 2, 5, 8, and 13 generally leads to decreased affinity across receptor subtypes

  • Position-specific effects include Pro5 substitution reducing hY2-receptor affinity 600-fold

  • Pro8 substitution decreasing hY4-receptor affinity 11-fold and hY5-receptor affinity 69-fold

• Truncated analogues: These can identify critical regions for receptor binding

  • Some truncated analogues show enhanced selectivity for specific receptor subtypes

• Competitive binding assays: Using labeled and unlabeled ligands to determine binding affinities and receptor densities

When designing such experiments, researchers should consider the rank order of affinity for pancreatic polypeptides (PP > PP (2-36) and [Ile-31, Gln-34] PP > [Pro-34] PYY > PYY and [Leu-31, Pro-34] NPY > NPY > PYY (3-36) and NPY (2-36) > PP (13-36) > PP (31-36) > NPY free acid) .

How can researchers validate NPY4R antibody specificity?

Validating NPY4R antibody specificity is crucial for reliable experimental results. Recommended approaches include:

• Positive control inclusion: Use cell lines with confirmed NPY4R expression (MCF7, SH-SY5Y, SW480, HT-29)

• Immunogen competition assays: Pre-incubate the antibody with the immunogen peptide (e.g., sequence corresponding to amino acids 1-80 of human NPY4R (NP_005963.4))

• Multiple antibody comparison: Use antibodies raised against different epitopes of NPY4R and compare detection patterns

• Knockout/knockdown controls: If available, include samples with NPY4R genetic deletion or suppression

• Cross-reactivity assessment: Test the antibody against related NPY receptors (Y1, Y2, Y5) to ensure specificity

Documentation of these validation steps strengthens the reliability of research findings and should be included in published methods.

What considerations are important when designing experiments to study NPY4R signaling pathways?

When investigating NPY4R signaling pathways, consider the following methodological aspects:

• Receptor coupling characteristics: NPY4R is negatively coupled to cAMP, so experimental readouts should include cAMP level measurements

• Ligand selection: Consider the differential affinity of various peptides (PP, NPY, PYY) for the receptor

• Post-translational modifications: Account for glycosylation and other modifications that may affect receptor function and antibody recognition

• Cellular localization: As a multi-pass membrane protein, proper membrane preparation protocols are essential for isolation and study

• Receptor internalization dynamics: Methods to track receptor trafficking (such as fluorescently labeled antibodies) may be required for comprehensive signaling studies

Incorporating these considerations will enhance experimental design and data interpretation in NPY4R signaling research.

What are the recommended protocols for NPY4R detection by Western blot?

For optimal NPY4R detection by Western blot:

• Sample preparation:

  • Use cell lines with confirmed NPY4R expression (e.g., MCF7, SH-SY5Y, SW480, HT-29)

  • Include appropriate membrane solubilization steps since NPY4R is a multi-pass membrane protein

• Antibody selection and dilution:

  • Use antibodies validated for Western blot, such as the CAB8143 polyclonal antibody

  • Apply recommended dilution ranges (1:500 - 1:2000 for CAB8143)

• Detection conditions:

  • Expected molecular weight for human NPY4R is approximately 42.2 kDa

  • Account for potential shifts due to post-translational modifications, particularly glycosylation

• Controls:

  • Include positive control samples from validated cell lines

  • Consider recombinant NPY4R protein as an additional control

• Troubleshooting:

  • For weak signals, optimize antibody concentration and incubation time

  • For multiple bands, verify whether they represent different glycosylation states or splice variants

Following these recommendations will improve detection specificity and results reliability.

What are the key considerations for immunohistochemical detection of NPY4R?

For successful immunohistochemical detection of NPY4R:

• Tissue preparation:

  • Optimal fixation is critical; overfixation may mask epitopes

  • Consider antigen retrieval methods for formalin-fixed tissues

• Antibody selection:

  • Choose antibodies validated for IHC applications

  • Consider the epitope location and accessibility in fixed tissues

• Detection strategy:

  • For fluorescent detection, minimize autofluorescence with appropriate quenching steps

  • For chromogenic detection, optimize development time to balance signal and background

• Controls:

  • Include tissues with known high (small intestine, colon) and low NPY4R expression

  • Consider peptide competition controls to verify specificity

• Interpretation:

  • Remember that NPY4R is a membrane-localized protein

  • Verify subcellular localization patterns against known distribution data

Careful attention to these factors will improve the reliability and interpretability of NPY4R immunohistochemical data.

How can researchers troubleshoot non-specific binding with NPY4R antibodies?

When encountering non-specific binding with NPY4R antibodies:

• Blocking optimization:

  • Test different blocking agents (BSA, normal serum, commercial blockers)

  • Increase blocking time or concentration if background persists

• Antibody dilution adjustment:

  • Titrate antibody to find optimal concentration that maximizes specific signal while minimizing background

  • Consider testing dilutions beyond the recommended range (1:500 - 1:2000)

• Wash protocol enhancement:

  • Increase wash duration and volume

  • Add detergent (0.05-0.1% Tween-20) to wash buffers if not already included

• Secondary antibody controls:

  • Include secondary-only controls to identify potential secondary antibody cross-reactivity

  • Consider switching secondary antibody suppliers if background persists

• Sample-specific considerations:

  • For highly autofluorescent tissues, consider chromogenic detection alternatives

  • For tissues with endogenous biotin, use biotin-free detection systems

These troubleshooting approaches can significantly improve signal-to-noise ratio in NPY4R detection experiments.

How can NPY4R antibodies be utilized in neuroscience research?

NPY4R antibodies offer valuable tools for neuroscience research:

• Receptor distribution mapping:

  • Immunostaining of neural tissues to map NPY4R expression patterns

  • Correlated with functional neuroanatomy to understand potential roles in neural circuits

  • Demonstrated application in rat dorsal root ganglion (DRG) neurons

• Receptor regulation studies:

  • Monitoring changes in NPY4R expression under various physiological or pathological conditions

  • Comparing receptor distribution in normal vs. disease states

• Co-localization analysis:

  • Double-labeling with markers for specific neuronal populations

  • Identification of NPY4R-expressing cells in heterogeneous neural tissues

• Drug development:

  • Screening potential compounds that modulate NPY4R activity

  • Evaluating receptor occupancy in response to candidate therapeutics

• Neural circuit investigation:

  • Identifying potential neuromodulatory influences via NPY4R activation

  • Understanding the role in stress response pathways

These applications provide critical insights into NPY4R's function in neural systems and potential therapeutic targeting.

What are the emerging applications of NPY4R antibodies in metabolic research?

NPY4R antibodies are increasingly valuable for metabolic research:

• Appetite regulation studies:

  • Detection of receptor expression in gut-brain axis components

  • Monitoring receptor levels in response to feeding/fasting states

  • Investigation of receptor expression in obesity models

• Enteroendocrine system analysis:

  • Localization of NPY4R in enteroendocrine cells

  • Correlation with hormone secretion patterns

  • Expression in tissues with known NPY4R presence (small intestine, colon)

• Pancreatic function research:

  • Given NPY4R's role as a pancreatic polypeptide receptor

  • Potential involvement in insulin/glucagon regulation pathways

• Stress-metabolism interaction studies:

  • Investigation of how stress signals modulate metabolic regulation through NPY4R pathways

  • Correlation with stress hormone levels

• Therapeutic target validation:

  • Confirmation of receptor accessibility in target tissues

  • Evaluation of receptor modulation by candidate compounds

These emerging applications highlight NPY4R's importance at the intersection of neural and metabolic regulation systems.

How do post-translational modifications affect NPY4R antibody recognition?

Post-translational modifications, particularly glycosylation, can significantly impact NPY4R antibody recognition :

• Epitope accessibility:

  • Glycosylation can mask antibody binding sites

  • Consider antibodies targeting non-glycosylated regions for consistent detection

• Molecular weight variations:

  • Western blot may reveal bands differing from the predicted 42.2 kDa size due to glycosylation

  • Multiple bands may represent differentially glycosylated forms rather than non-specific binding

• Deglycosylation experiments:

  • Enzymatic deglycosylation (PNGase F, Endo H) can confirm glycosylation status

  • Comparison of treated and untreated samples may clarify antibody recognition patterns

• Antibody selection strategy:

  • For studies focusing on total NPY4R levels, choose antibodies targeting conserved epitopes less affected by modifications

  • For studying specific modified forms, select antibodies with documented sensitivity to modification state

Understanding these effects is crucial for accurate interpretation of experimental results and proper antibody selection.

What considerations are important when designing quantitative assays for NPY4R?

When developing quantitative assays for NPY4R:

• Standard curve development:

  • Use recombinant NPY4R fragments or full-length protein

  • Validate linear range and detection limits

• Sample preparation standardization:

  • Establish consistent protocols for membrane protein extraction

  • Normalize to total protein or housekeeping proteins for comparative analysis

• Antibody validation:

  • Confirm antibody specificity using positive controls (MCF7, SH-SY5Y, SW480, HT-29)

  • Determine optimal antibody concentration for quantitative reliability

• Assay format selection:

  • ELISA for quantification of NPY4R in solution

  • Quantitative Western blot for relative expression comparisons

  • Flow cytometry for cell surface expression quantification

• Data normalization strategy:

  • Account for background signal

  • Include internal standards for inter-assay comparisons

Attention to these factors will enhance the reproducibility and reliability of quantitative NPY4R measurements.