NPBWR2 Antibody
Description
Introduction to NPBWR2 Antibody
The NPBWR2 antibody is a specialized immunological tool designed to detect the human neuropeptide B/W receptor type 2 (NPBWR2), a G-protein coupled receptor (GPCR) expressed in discrete brain regions and peripheral tissues. This antibody facilitates the study of NPBWR2’s role in physiological processes such as pain modulation, energy homeostasis, and emotional regulation .
Structure and Function of NPBWR2
NPBWR2, encoded by the NPBWR2 gene on chromosome 20q13.3, shares 64% sequence homology with its paralog, NPBWR1. Both receptors bind neuropeptides B (NPB) and W (NPW) with varying affinities, though NPBWR2 exhibits a potency rank order of NPW23 > NPW30 > NPB . The receptor couples to Gi-class G-proteins, inducing inhibitory neuronal signaling via GIRK channels . Key structural features include seven transmembrane domains and conserved amino acid motifs similar to opioid and somatostatin receptors .
| Characteristic | NPBWR2 |
|---|---|
| Gene Location | Chromosome 20q13.3 |
| Protein Length | 333 amino acids |
| Sequence Homology | 64% with NPBWR1 |
| Ligands | NPB, NPW (NPW23 > NPW30 > NPB) |
| G-Protein Coupling | Gi-class |
3.1. Pain Modulation
Immunofluorescence studies in rat heart and dorsal root ganglia (DRG) revealed NPBWR2 expression in cardiomyocytes and nerve fibers, suggesting a role in chronic inflammatory pain regulation . Pre-adsorption assays confirmed antibody specificity for NPBWR2 in rat tissues .
3.2. Fibroblast Signaling
In human dermal fibroblasts, RT-qPCR detected NPBWR2 mRNA at 32-fold higher levels than NPBWR1. Immunofluorescence confirmed receptor presence on mesenchymal cells, though Western blot faced challenges due to antibody specificity .
3.3. Neuroendocrine Regulation
NPBWR2 is expressed in the amygdala and hippocampus, with studies linking its polymorphisms to emotional responses . Antibody-based assays have mapped receptor distribution in brain regions implicated in fear and anxiety .
Challenges in Detection
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Antibody Specificity: Western blot failures in human fibroblasts highlight the need for optimized protocols or alternative methods like mass spectrometry .
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Tissue Variability: Absence in rodent genomes complicates cross-species studies .
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Low Expression Levels: Fibroblast studies required high-sensitivity assays to detect NPBWR1/2 .
Product Specs
Constituents: 50% Glycerol, 0.01M PBS, pH 7.4
Target Background
- Primarily expressed in the brain or pituitary. PMID: 27399877
Q&A
How should researchers validate NPBWR2 antibody specificity in human brain tissue?
Validation requires a multi-step approach:
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Positive/Negative Controls: Use human brain regions with known NPBWR2 expression (e.g., amygdala, hippocampus) and tissues lacking NPBWR2 (e.g., rodent brain) .
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Knockout Validation: CRISPR/Cas9-mediated NPBWR2 knockout cell lines can confirm antibody specificity, though primate models are required due to rodent absence .
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Orthogonal Techniques: Pair immunohistochemistry (IHC) with RNAscope® in situ hybridization to correlate protein and mRNA localization (e.g., human pituitary corticotrophs) .
| Validation Parameter | Recommended Protocol |
|---|---|
| Antigen Retrieval | TE buffer pH 9.0 or citrate buffer pH 6.0 |
| Dilution Range | IHC: 1:50–1:500 |
| Target Confirmation | Co-staining with ACTH in pituitary cells |
What experimental applications are NPBWR2 antibodies suitable for?
NPBWR2 antibodies are primarily validated for:
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IHC: Localization in formalin-fixed paraffin-embedded (FFPE) human brain sections (caudate nucleus, hippocampus) .
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ELISA: Quantification in pituitary homogenates under stress-axis studies .
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Immunofluorescence (IF): Co-localization with dopaminergic markers (tyrosine hydroxylase) in midbrain projections .
Critical Consideration: NPBWR2’s multi-pass membrane structure necessitates Triton X-100 permeabilization (0.1–0.3%) for epitope accessibility .
How does tissue fixation affect NPBWR2 antibody performance?
Prolonged formalin fixation (>24 hr) masks NPBWR2 epitopes. Optimal protocols include:
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Antigen Retrieval: Heat-induced epitope retrieval (HIER) with TE buffer restores 85–90% signal in FFPE sections .
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Post-Fixation Treatment: 1 hr incubation in 0.1 M glycine to reduce aldehyde crosslinking .
How to resolve contradictory NPBWR2 expression data across studies?
Discrepancies often arise from:
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Species Differences: NPBWR2 is absent in rodents but detected in primates and chickens . Studies using rodent models may report false positives due to NPBWR1 cross-reactivity .
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Antibody Clones: Polyclonal antibodies (e.g., Proteintech 24162-1-AP) show broader reactivity than monoclonals. Validate using recombinant NPBWR2-transfected HEK293 cells .
What multiplex assays are recommended for studying NPBWR2 signaling?
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cAMP Inhibition Assays: NPBWR2 activation reduces forskolin-induced cAMP by 60–70% in CHO-K1 cells (EC₅₀: 15.8 nM for NPB) .
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Calcium Imaging: Co-transfect NPBWR2 with chimeric Gαq proteins to assess ligand-dependent Ca²⁺ flux .
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Phospho-ERK Profiling: NPBWR2 activation increases pERK1/2 in adrenal carcinoma cells via β/γ G-protein subunits .
Protocol Note: Use 100 nM NPW23 for maximal receptor activation in human cell lines .
How to investigate NPBWR2 ligand-receptor interactions in vivo?
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Ligand Specificity: NPW23 shows higher affinity for NPBWR2 (EC₅₀: 2.1 nM) versus NPBWR1 (EC₅₀: 0.23 nM) .
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Genetic Models: Use NPBWR2-overexpressing zebrafish for stress-axis studies, as their pituitary anatomy permits high-resolution imaging .
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Behavioral Assays: Pair intracranial NPW injections with elevated plus maze tests in primates to quantify anxiety-like behaviors .
What genetic tools address NPBWR2’s absence in rodent models?
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Primate Models: Rhesus macaques express NPBWR2 in the amygdala and hippocampus .
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Avian Systems: Chickens exhibit NPBWR2 in pituitary corticotrophs, enabling study of glucocorticoid feedback .
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Transgenic Mice: Humanized NPBWR2 knock-in mice show 78% receptor homology but require immune tolerance induction .
How to quantify NPBWR2 in heterogenous cell populations?
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Flow Cytometry: Use Pacific Blue-conjugated antibodies (1:100) on dissociated pituitary cells .
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Spatial Transcriptomics: Combine MERFISH with IHC to map NPBWR2⁺ cells in human amygdala subnuclei .
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LC-MS/MS: Quantify tryptic peptides (e.g., VVLLFIAQR) from immunoprecipitated NPBWR2 .
What confounds NPBWR2 antibody performance in Western blotting?
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Glycosylation: NPBWR2’s 37 kDa core migrates at 45–50 kDa in SDS-PAGE due to N-linked glycosylation .
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Proteolytic Degradation: Add 1 μM leupeptin during homogenization to preserve full-length receptors .
| Migration Anomaly | Troubleshooting Step |
|---|---|
| Multiple Bands | Deglycosylate lysates with PNGase F (37°C, 1 hr) |
| Smear Artifacts | Use 4–12% Bis-Tris gels with MOPS buffer |
How does NPBWR2 crosstalk with other GPCRs?
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D2 Dopamine Receptors: NPBWR2⁺ neurons in the CeAL co-express D2R, enabling synergistic inhibition of cAMP .
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Somatostatin Receptors: Co-activation reduces GH secretion by 40% in chicken pituitary via Gi/o coupling .
Experimental Design: Use BRET assays to quantify NPBWR2-D2R heterodimerization in live cells .
