NPFFR2 Antibody

What is NPFFR2 and what are its primary biological functions?

NPFFR2 (Neuropeptide FF Receptor 2), also known as GPR74, NPFF2, or Neuropeptide G-protein coupled receptor, is a receptor for NPAF (A-18-F-amide) and NPFF (F-8-F-amide) neuropeptides, which are also referred to as morphine-modulating peptides. This receptor mediates its action through association with G proteins that activate a phosphatidylinositol-calcium second messenger system .

NPFFR2 has diverse physiological roles, including regulation of pain modulation, metabolism, and more recently discovered involvement in cancer progression. It functions as a crucial regulator of diet-induced adaptive thermogenesis in mice . The receptor's activation inhibits adenylate cyclase in cells, as demonstrated by its ability to strongly inhibit forskolin-induced cAMP increases in human arcuate-like neurons (hALNs) with an IC50 of 0.1 ± 0.01 nM . Additionally, emerging research has identified NPFFR2 as a contributor to hepatocellular carcinoma (HCC) malignancy through RhoA/YAP pathway enhancement .

What experimental applications are NPFFR2 antibodies most commonly used for?

NPFFR2 antibodies are employed across various experimental applications, with immunohistochemistry on paraffin-embedded tissues (IHC-P) being a primary validated application for commercial antibodies like the rabbit polyclonal ab140900 . Based on current research practices, these antibodies serve critical functions in:

• Tissue expression profiling: Detecting NPFFR2 expression across normal and pathological tissues, particularly in comparative studies between normal liver and hepatocellular carcinoma samples .

• Cellular localization studies: Confirming protein expression and subcellular distribution in cell lines, as demonstrated in HCC cell lines through immunostaining .

• Validation of knockdown experiments: Verifying the efficiency of NPFFR2 depletion in siRNA experiments through immunostaining or Western blotting .

• Co-expression analysis: Investigating the co-expression of NPFFR2 with other proteins, such as the co-expression of Npffr2 with Npy and Agrp in mouse arcuate nucleus neurons .

Each application requires specific optimization protocols depending on the experimental model and tissue type being investigated.

How is NPFFR2 expression distributed across different tissues and cell types?

NPFFR2 shows distinct expression patterns across tissues and cell types, with significant implications for both physiological function and pathological states:

In the central nervous system, NPFFR2 expression has been confirmed in the human arcuate nucleus (ARC) . Single-cell gene expression analyses in mice revealed that Npffr2+ neurons predominantly co-express Npy and Agrp in relatively high abundance (3.02 ± 0.21 and 2.31 ± 0.17, respectively) . These neurons are also characterized by GABAergic, glutaminergic, and dopaminergic gene expression profiles, albeit to a lower degree .

In cancer contexts, TCGA dataset analysis through the UALCAN platform revealed elevated NPFFR2 expression in HCC compared to normal tissues, with expression levels correlating with tumor grade and nodal metastasis status . This differential expression pattern underscores NPFFR2's potential role as both a biomarker and therapeutic target in cancer research.

What are the optimal conditions for using NPFFR2 antibodies in immunohistochemistry?

When using NPFFR2 antibodies for immunohistochemistry on paraffin-embedded tissues (IHC-P), researchers should consider the following optimized protocol based on validated antibodies like ab140900 :

• Tissue preparation: Use formalin-fixed, paraffin-embedded tissue sections at 4-6 μm thickness.

• Antigen retrieval: Perform heat-mediated antigen retrieval using citrate buffer (pH 6.0) to expose epitopes that may be masked during fixation.

• Blocking and permeabilization: Block with 5-10% normal serum from the same species as the secondary antibody for 1 hour at room temperature. Include 0.1-0.3% Triton X-100 for membrane permeabilization.

• Primary antibody incubation: Dilute NPFFR2 antibody to the manufacturer's recommended concentration (typically 1:100 to 1:500 for polyclonal antibodies) and incubate overnight at 4°C.

• Detection system: Use a biotin-free polymer detection system to minimize background staining, especially important when working with liver tissues that may have endogenous biotin.

• Controls: Always include positive controls (tissues known to express NPFFR2, such as certain hypothalamic regions) and negative controls (primary antibody omission and isotype controls) to validate staining specificity.

• Counterstaining: Use hematoxylin for nuclear counterstaining, but keep staining time brief to avoid obscuring positive signals, particularly in tissues with high NPFFR2 expression like HCC samples .

For dual immunofluorescence studies examining co-expression with other markers, additional optimization may be required to ensure antibody compatibility and minimize cross-reactivity.

How should researchers validate the specificity of NPFFR2 antibodies?

Comprehensive validation of NPFFR2 antibody specificity is essential to ensure reliable experimental outcomes. Recommended validation approaches include:

• Peptide competition assays: Pre-incubate the antibody with excess synthetic peptide used as immunogen to confirm signal suppression in positive samples.

• Genetic knockdown validation: Verify reduction in antibody signal following siRNA-mediated NPFFR2 depletion, as demonstrated in HCC cell line studies where both mRNA expression and protein levels were assessed after knockdown .

• Overexpression controls: Confirm increased signal in cells overexpressing NPFFR2 through transient transfection compared to empty vector controls .

• Western blot analysis: Validate antibody specificity by confirming a single band of appropriate molecular weight in positive samples and absence in negative controls.

• Cross-reactivity assessment: Test antibody against related receptors (particularly NPFFR1) to ensure specificity, as these receptors share structural similarities but are generally not co-localized in the same neurons .

• Correlation of protein and mRNA data: Compare antibody staining patterns with mRNA expression data (from RT-PCR, RNA-seq, or in situ hybridization) to confirm concordance across methodologies .

A validation matrix documenting these specificity controls should be maintained for each new lot of antibody to ensure experimental reproducibility and data reliability.

What controls should be included when using NPFFR2 antibodies in functional studies?

Robust experimental design for functional studies using NPFFR2 antibodies requires multiple control types:

• Technical controls:

  • Antibody concentration gradient: Titrate antibody dilutions to determine optimal signal-to-noise ratio

  • Incubation time comparison: Test different incubation periods to maximize specific binding while minimizing background

  • Blocking optimization: Evaluate different blocking reagents to address tissue-specific background issues

• Biological controls:

  • Positive controls: Include tissues with known NPFFR2 expression, such as hypothalamic sections or NPFFR2-expressing cell lines

  • Negative controls: Use tissues from knockout models or cell lines with confirmed absence of NPFFR2 expression

  • Receptor activation controls: Include parallel experiments with NPFF ligand stimulation (3 μM is effective in functional assays) to confirm receptor functionality

• Functional validation controls:

  • Parallel techniques: Validate findings using complementary approaches (e.g., combine immunostaining with functional assays like cAMP measurement)

  • Rescue experiments: Restore NPFFR2 expression in knockdown cells to confirm phenotype reversal

  • Dose-response relationships: Establish dose-dependent effects of NPFFR2 modulation, as demonstrated in cAMP inhibition studies showing an IC50 of 0.1 ± 0.01 nM for NPFF in hALNs

How can researchers investigate NPFFR2-mediated signaling pathways using antibody-based approaches?

Investigating NPFFR2-mediated signaling requires integrating antibody-based approaches with functional assays to delineate both proximal and distal signaling events:

• G protein coupling assessment:

  • Use co-immunoprecipitation with NPFFR2 antibodies followed by immunoblotting for various G protein subunits to identify coupling partners

  • Complement with functional assays targeting second messengers (cAMP inhibition has been demonstrated with an IC50 of 0.1 ± 0.01 nM in hALNs)

  • Consider structural biology approaches similar to those used in elucidating the hNPSF–NPFFR2–Gi complex

• Downstream effector analysis:

  • Employ phospho-specific antibodies to key signaling intermediates to track pathway activation kinetics

  • In HCC research, focus on RhoA signaling components, as NPFFR2 increases invasiveness through RhoA activation

  • Investigate YAP activity modulation, as NPFFR2-mediated regulation of RhoA affects YAP activity and F-actin formation

• Receptor trafficking and internalization:

  • Use surface biotinylation combined with NPFFR2 immunoprecipitation to track receptor internalization kinetics

  • Perform time-course immunofluorescence studies with NPFFR2 antibodies to visualize receptor redistribution following ligand stimulation

  • Consider pulse-chase experiments with differentially labeled antibodies to distinguish surface from internalized receptor pools

• Signaling crosstalk investigation:

  • Use dual immunofluorescence to co-localize NPFFR2 with other receptors or signaling components

  • Perform proximity ligation assays with NPFFR2 antibodies paired with antibodies to putative interaction partners

  • Investigate the intersection between NPFFR2 and opioid receptor signaling, given NPFF's role as a morphine-modulating peptide

These methodological approaches leverage antibody specificity to unravel complex signaling cascades initiated by NPFFR2 activation.

How are NPFFR2 antibodies being utilized in cancer research, particularly in hepatocellular carcinoma studies?

NPFFR2 antibodies have become instrumental in cancer research, with significant applications in hepatocellular carcinoma (HCC) studies:

• Expression profiling and prognostic evaluation:

  • NPFFR2 antibodies are used in tissue microarrays to correlate expression with clinical outcomes

  • IHC studies have revealed that NPFFR2 is overexpressed in HCC tissues compared to normal liver, with expression levels correlating with poor prognosis

  • Analysis of TCGA data has confirmed elevated expression across tumor grades and in nodal metastasis

• Functional phenotyping in cancer models:

  • NPFFR2 antibodies validate expression changes in gain-of-function and loss-of-function experimental models

  • Immunostaining confirms protein reduction in siRNA-mediated knockdown experiments in HCC cell lines

  • Expression verification in cell lines used for functional assays investigating invasion, migration, and clonogenic survival

• Mechanistic pathway investigation:

  • Paired with phospho-specific antibodies to investigate RhoA/YAP pathway activation

  • Used in studies demonstrating that NPFFR2 increases invasiveness and migration in HCC cells through RhoA signaling

  • Applied in research showing that NPFFR2 regulation of RhoA affects F-actin formation and YAP activity

• Therapeutic target validation:

  • Employed in drug screening studies to identify compounds that modulate NPFFR2 expression or function

  • Combined with structural biology approaches to develop more selective NPFFR2-targeting drugs with potentially improved therapeutic profiles compared to existing multitarget ligands like BN-9 and DN-9

The multifaceted application of NPFFR2 antibodies in cancer research highlights their value beyond simple detection, extending to mechanistic insights and therapeutic development.

What approaches can be used to study NPFFR2's structural characteristics and ligand binding?

Investigating NPFFR2's structural characteristics and ligand binding involves multidisciplinary approaches combining antibody-based techniques with advanced structural biology methods:

• Cryo-electron microscopy (cryo-EM) studies:

  • Recent research has successfully employed single-particle cryo-EM to elucidate the structure of human NPSF-bound NPFFR2 in complex with Gi

  • This approach revealed that the hydrophilicity of the ligand-binding pocket, encompassing amino acids at the 5th and 6th positions from the C-terminus of human NPSF, is critical for ligand selectivity

  • Structural comparison between inactive and active states provides insights into the TM3-mediated activation mechanism of NPFFR2 upon ligand binding

• Antibody-facilitated structural stabilization:

  • For the ligand-free state structure of NPFFR2, researchers replaced residues 246-266 in the ICL3 region with cytochrome b562 RIL (BRIL) and purified the receptor in complex with anti-BRIL Fab and anti-Fab nanobody

  • This approach stabilized the complex for cryo-EM analysis, providing a global map at 2.9 Å resolution and a local resolution map of the receptor at 3.6 Å

• Binding site mapping:

  • Site-directed mutagenesis combined with functional assays (e.g., cAMP inhibition) to identify critical residues for ligand binding

  • Homology modeling based on crystal structures, as demonstrated for NPFFR1

  • Antibody-based protection assays to identify surface-exposed epitopes involved in ligand binding

• Conformational dynamics investigation:

  • Study the distinct conformational changes induced by different ligands (NPFF, NPAF, and hNPSF)

  • Investigate the structural basis for the observation that hNPSF (which contains additional SQA residues at the N-terminus of NPFF) and NPAF exhibit enhanced potency compared to NPFF

  • Explore how binding of the RF-amide motif in the TMD pocket triggers TM3-mediated conformational changes leading to receptor activation

These approaches collectively provide complementary insights into NPFFR2 structure-function relationships, with important implications for drug design targeting this receptor.

How can researchers address non-specific binding of NPFFR2 antibodies?

Non-specific binding is a common challenge with NPFFR2 antibodies that can compromise data interpretation. Researchers can implement several strategies to minimize this issue:

• Optimization of blocking conditions:

  • Test multiple blocking agents (BSA, serum, commercial blocking buffers) at various concentrations

  • Consider tissue-specific blockers (e.g., liver powder for liver tissue studies) to absorb cross-reactive antibodies

  • Extend blocking time to 2 hours at room temperature for difficult samples

• Antibody dilution optimization:

  • Perform systematic titration series to identify the optimal antibody concentration that maximizes specific signal while minimizing background

  • For polyclonal antibodies like ab140900, starting dilutions of 1:100-1:500 are recommended, with further optimization based on signal-to-noise ratio

• Pre-absorption techniques:

  • Pre-absorb antibodies with the immunogen peptide as a specificity control

  • For polyclonals raised against synthetic peptides within human NPFFR2, pre-absorption with this peptide should eliminate specific staining

  • Consider cross-absorption with related receptor peptides (particularly NPFFR1) to remove antibodies that might recognize shared epitopes

• Washing protocol modification:

  • Implement more stringent washing conditions (increased detergent concentration, longer wash times)

  • Use high-salt washes (up to 500 mM NaCl) to disrupt low-affinity non-specific interactions

  • Consider adding detergent concentrations in a gradient across wash steps

• Secondary antibody selection:

  • Choose highly cross-adsorbed secondary antibodies to minimize species cross-reactivity

  • Test multiple secondary antibody formats (direct conjugates vs. detection systems) to identify optimal signal-to-noise ratio

  • Consider using Fab or F(ab')2 fragments instead of whole IgG to reduce Fc-mediated binding to tissue

Implementation of these approaches should be systematically documented to identify the combination that yields optimal results for specific experimental conditions.

How should contradictory data regarding NPFFR2 expression or function be interpreted?

When confronted with contradictory data regarding NPFFR2 expression or function, researchers should implement a systematic analytical framework:

• Methodological comparison:

  • Evaluate the detection methods used (antibody-based vs. mRNA-based techniques)

  • Consider that RNA-protein correlation for NPFFR2 may not be perfect due to post-transcriptional regulation

  • Assess sensitivity differences between methods (e.g., RT-PCR may detect expression below the threshold for immunohistochemical detection)

• Sample heterogeneity assessment:

  • NPFFR2 expression varies across tissues and cell types, with heterogeneous expression even within the same tissue type

  • Consider cell-type specific expression patterns, particularly in complex tissues like the hypothalamus where NPFFR2 is primarily expressed in specific neuronal populations

  • Analyze single-cell data when available to account for cellular heterogeneity

• Experimental condition analysis:

  • Regulatory mechanisms may affect NPFFR2 expression under different experimental conditions

  • In cancer models, differences may reflect the varied origins and genetic backgrounds of cell lines

  • Consider dynamic regulation of NPFFR2 in response to environmental stimuli or disease progression

• Species-specific differences:

  • Human and mouse NPFFR2 show important structural and functional differences

  • Cross-species comparison should account for these differences when interpreting contradictory findings

  • Recent structural studies have highlighted species-specific characteristics in receptor-ligand interactions

• Integrated data analysis approach:

  • Implement multimodal validation combining complementary techniques

  • Consider hierarchical data weighting based on methodological rigor

  • Develop consensus interpretation models that accommodate apparently contradictory findings

What are common pitfalls in NPFFR2 antibody-based research and how can they be avoided?

Several common pitfalls can compromise NPFFR2 antibody-based research. Awareness and proactive strategies can help researchers avoid these issues:

By anticipating these common pitfalls and implementing the suggested solutions, researchers can significantly enhance the reliability and impact of their NPFFR2 antibody-based research.

What are the future directions for NPFFR2 antibody research?

The evolving landscape of NPFFR2 research presents several promising future directions for antibody-based investigations:

• Development of conformation-specific antibodies: Creating antibodies that selectively recognize active versus inactive conformations of NPFFR2 would enable spatiotemporal tracking of receptor activation states in situ. Recent structural elucidation of both ligand-free and ligand-bound NPFFR2 conformations provides the foundation for designing such tools .

• Integration with advanced imaging technologies: Combining NPFFR2 antibodies with super-resolution microscopy or expansion microscopy will provide unprecedented insights into receptor nanoscale organization and dynamics in both healthy and pathological contexts.

• Single-cell analysis applications: Developing antibody-based protocols compatible with single-cell protein analysis will complement existing single-cell transcriptomic data on NPFFR2 expression, addressing the current gap in human hypothalamic neuron characterization .

• Therapeutic development facilitation: NPFFR2 antibodies will play crucial roles in screening and validating novel therapeutic compounds targeting this receptor. The structural understanding of NPFFR2 now opens opportunities to develop drugs with enhanced receptor subtype selectivity compared to existing multitarget ligands like BN-9 and DN-9 .

• Cancer biomarker exploration: Further investigation of NPFFR2 as a potential prognostic or predictive biomarker in hepatocellular carcinoma and potentially other cancer types, building on findings of its overexpression in HCC and correlation with poor prognosis .

These future directions highlight the continued importance of high-quality, well-validated NPFFR2 antibodies in advancing our understanding of this receptor's complex biology and therapeutic potential across multiple disease contexts.

How does recent structural research impact NPFFR2 antibody applications?

Recent structural research on NPFFR2, particularly the elucidation of the hNPSF–NPFFR2–Gi complex structure, significantly impacts antibody applications in several ways:

• Epitope-specific antibody development: The detailed structural information now available allows for rational design of antibodies targeting specific functional domains of NPFFR2. This knowledge enables the creation of antibodies that can selectively interfere with or detect particular receptor-ligand or receptor-effector interactions .

• Conformational state discrimination: Understanding the structural differences between ligand-free and ligand-bound states of NPFFR2 provides the basis for developing antibodies that can distinguish between these conformational states, offering powerful tools for studying receptor activation dynamics in situ .

• Validation tool refinement: Structural insights can guide more precise validation of existing antibodies by mapping their epitopes relative to functionally important domains, enhancing confidence in experimental interpretations.

• Cross-reactivity prediction: The homology model comparing NPFFR1 and NPFFR2 structures helps predict potential cross-reactivity of antibodies between these related receptors, informing antibody selection for subtype-specific studies .

• Drug development applications: Antibodies targeting specific structural features of NPFFR2 can serve as tools in drug screening assays, particularly for identifying compounds that selectively modulate NPFFR2 over NPFFR1, addressing the challenges of cross-reactivity seen with existing multitarget ligands .