NPFFR2 Antibody, HRP conjugated

What is NPFFR2 and why is it an important research target?

NPFFR2 (Neuropeptide FF Receptor 2) is a G protein-coupled receptor that belongs to the G-protein coupled receptor 1 family. It functions as a receptor for NPAF (A-18-F-amide) and NPFF (F-8-F-amide) neuropeptides, which are also known as morphine-modulating peptides . NPFFR2 has gained significant research interest due to its involvement in:

• Pain modulation and analgesia pathways

• Opioid system regulation

• Regulation of metabolic processes

• Potential role in cancer progression, particularly hepatocellular carcinoma

• Immune system regulation, particularly in macrophage function

Recent research has demonstrated that NPFFR2 is significantly upregulated in liver cancer, and its expression correlates with poor prognosis, making it a valuable target for both diagnostic and therapeutic development .

What applications are NPFFR2 antibodies, HRP conjugated suitable for?

For optimal results, the working dilution should be determined empirically for each experimental setup .

How should NPFFR2 antibody, HRP conjugated be stored to maintain activity?

To maintain optimal activity of the NPFFR2 antibody, HRP conjugated, follow these evidence-based storage recommendations:

• Store at -20°C in a non-frost-free freezer

• Ensure storage in aliquots to avoid repeated freeze/thaw cycles which can degrade antibody activity

• Most preparations are supplied in PBS buffer with 0.02% sodium azide and 50% glycerol at pH 7.3

• Some formulations contain 0.03% Proclin-300 as a preservative

• Properly stored, the antibody remains stable for approximately one year after shipment

• Do not cryopreserve the sealed kit components as indicated by manufacturers

For research involving long-term storage, validation of antibody activity is recommended before critical experiments.

What are the typical specificity characteristics of NPFFR2 antibodies?

NPFFR2 antibodies demonstrate specific binding characteristics that should be considered when designing experiments:

• The HRP-conjugated NPFFR2 antibody (ABIN7139946) specifically targets amino acids 23-43 of human NPFFR2

• Polyclonal antibodies generated in rabbits typically recognize multiple epitopes of the target protein

• High-quality preparations show >95% purity following Protein G purification

• Many commercial antibodies can recognize multiple isoforms of NPFFR2 (isoform1, isoform2, and isoform3)

• Cross-reactivity is primarily observed with human samples, though some antibodies may cross-react with samples from horse, cow, or monkey depending on the epitope targeted

Validation of antibody specificity in your experimental system is essential, particularly when working with novel tissue types or species.

How should sandwich ELISA be optimized for NPFFR2 detection using HRP-conjugated antibodies?

For optimal NPFFR2 detection using sandwich ELISA with HRP-conjugated antibodies, follow this methodological approach:

• Microplate preparation: Use plates pre-coated with anti-NPFFR2 capture antibody targeting a different epitope than your detection antibody

• Sample preparation:

  • For serum/plasma: Dilute samples appropriately (typically 1:2 to 1:10) in sample diluent

  • For cell culture supernatants: May be used undiluted or diluted depending on expected concentration

  • For cell/tissue lysates: Prepare in RIPA or similar buffer with protease inhibitors

• Protocol optimization:

  • Add 100μL of standards or properly diluted samples to wells

  • Incubate at room temperature (typically 90-120 minutes)

  • Wash plates thoroughly (3-5 times)

  • Add 100μL of biotinylated detection antibody

  • Incubate (60 minutes)

  • Wash thoroughly

  • Add HRP-Streptavidin Conjugate (SABC) and incubate (30 minutes)

  • Wash thoroughly

  • Add TMB substrate solution and monitor color development

  • Stop reaction with 1M HCl when appropriate

• Data analysis:

  • Read absorbance at 450nm

  • Generate standard curve using appropriate regression model

  • Calculate NPFFR2 concentration in samples from standard curve

The detection range for NPFFR2 typically spans 0.625-40ng/ml with a sensitivity threshold of approximately 0.375ng/ml .

What controls are essential when using NPFFR2 antibody, HRP conjugated in experimental research?

Implementing appropriate controls is crucial for obtaining reliable results with NPFFR2 antibody, HRP conjugated:

• Positive controls:

  • Human small intestine tissue sections (for IHC applications)

  • Human placenta or testis tissue (alternative positive controls)

  • Cell lines with confirmed NPFFR2 expression (e.g., certain hepatocellular carcinoma cell lines like Huh7)

• Negative controls:

  • Isotype control (rabbit IgG at equivalent concentration)

  • Secondary antibody-only control

  • Tissues/cells known not to express NPFFR2

  • Competitive blocking with immunizing peptide

• Technical controls:

  • Standard curve with recombinant NPFFR2 protein

  • Internal reference protein (housekeeping gene/protein)

  • Signal specificity validation using NPFFR2 knockdown/knockout samples

• Antibody validation controls:

  • ELISA-based surface expression assay as described in recent studies

  • Western blot confirmation of specificity at expected molecular weight (~60 kDa)

These controls should be customized based on your specific experimental design and the biological questions being addressed.

How can researchers distinguish between NPFFR1 and NPFFR2 in experimental systems?

Distinguishing between the closely related receptors NPFFR1 and NPFFR2 requires careful methodological considerations:

• Antibody selection strategies:

  • Use antibodies targeting non-conserved regions between the receptors

  • NPFFR2 antibodies targeting amino acids 23-43 or the C-terminal region provide higher specificity

  • Validate antibody specificity using overexpression systems or knockout controls

• Functional discrimination approaches:

  • Selective agonists: NPFF peptides preferentially activate NPFFR2 while RFRP peptides preferentially activate NPFFR1

  • Differential responses: NPFFR2 is strongly activated by neuropeptides FF (NPFFs) but shows low activity to RF-amide-related peptides (RFRPs)

  • Key amino acid differences: NPFFs contain Phe-Gln sequences at positions 5-6 from C-terminus, while RFRPs contain Asn-Leu at these positions

• Molecular techniques for discrimination:

  • RT-PCR with receptor-specific primers

  • Receptor-specific siRNA knockdown validation

  • Expression pattern analysis (NPFFR2 is upregulated in hepatocellular carcinoma)

• Structural considerations:

  • Recent structural studies (2025) have revealed that the C-terminal RF-amide moiety engages conserved residues in the transmembrane domain, while the N-terminal segment interacts in a receptor subtype-specific manner

  • This structural knowledge can inform the design of discriminatory experiments

What sample preparation methods are recommended for NPFFR2 detection in different tissue types?

Optimal sample preparation for NPFFR2 detection varies by tissue type and experimental approach:

• Cell lysate preparation:

  • Adherent cells: Wash with cold PBS, add RIPA buffer with protease inhibitors (1mL per 10^7 cells)

  • Suspension cells: Centrifuge, wash pellet with PBS, add lysis buffer

  • Homogenize by sonication or needle passage (3-5 cycles)

  • Centrifuge at 14,000g for 15 minutes at 4°C and collect supernatant

  • Determine protein concentration using BCA assay

• Tissue preparation for IHC:

  • Fix tissues in 4% paraformaldehyde

  • Process and embed in paraffin

  • Section at 4-6μm thickness

  • For NPFFR2 detection, antigen retrieval with TE buffer (pH 9.0) is recommended, though citrate buffer (pH 6.0) may also be used

  • Block with 5% BSA for 1-2 hours before antibody application

• Serum/plasma processing:

  • Collect blood in appropriate tubes (EDTA for plasma, activator tubes for serum)

  • Centrifuge within 30 minutes of collection (1,500g for 15 minutes)

  • Aliquot to avoid freeze-thaw cycles

  • For NPFFR2 detection, dilution ratios typically range from 1:2 to 1:10 in assay buffer

• Bone marrow-derived macrophages:

  • For studies investigating NPFFR2 in immune cells, harvest bone marrow cells from femurs and tibias

  • Culture in DMEM containing 10% FBS and 20% L929 cell-conditioned medium for 7 days

  • Confirm macrophage differentiation by flow cytometry using F4/80 and CD11b antibodies

How does NPFFR2 signaling impact downstream molecular pathways in hepatocellular carcinoma?

Recent research has elucidated the complex signaling mechanisms through which NPFFR2 contributes to hepatocellular carcinoma (HCC) progression:

• Key signaling pathway interactions:

  • NPFFR2 activates RhoA/YAP signaling pathway in HCC cells

  • NPFFR2-mediated RhoA activation leads to enhanced F-actin formation

  • Downstream YAP nuclear translocation promotes transcription of pro-tumorigenic genes

  • Inhibition of Rho kinase activity completely restores phenotypes induced by NPFFR2

• Molecular mechanisms and experimental evidence:

  • RhoA activity assessment in Huh7 cells transfected with NPFFR2 showed increased activation

  • Experimental validation using RhoA Activation Assay Kit (Cytoskeleton, Inc.)

  • Western blot detection confirmed RhoA-mediated signaling activation

  • Immunofluorescence imaging with phalloidin staining demonstrated increased F-actin formation

• Functional consequences in HCC:

  • NPFFR2 silencing reduced malignancy by decreasing cell survival, invasion, and migration

  • NPFFR2 overexpression increased invasion, migration, and anchorage-independent growth

  • Expression of NPFFR2 in HCC tissues (75% of samples) correlated with poor prognosis

  • The data suggest NPFFR2 as a potential therapeutic target for HCC treatment

These findings provide a mechanistic framework for developing targeted therapeutics against NPFFR2 in HCC patients.

What structural insights have been revealed about NPFFR2 and how do they inform antibody development?

Recent cryo-electron microscopy (cryo-EM) studies have provided groundbreaking insights into NPFFR2 structure:

These structural insights provide a foundation for developing next-generation antibodies with enhanced specificity and functional properties.

How can researchers address the challenge of detecting low NPFFR2 expression in normal tissues versus overexpression in pathological conditions?

Detecting differential NPFFR2 expression between normal and pathological tissues requires sophisticated methodological approaches:

• Enhanced detection sensitivity strategies:

  • Signal amplification using tyramide signal amplification (TSA) for IHC applications

  • Employ droplet digital PCR (ddPCR) for absolute quantification of low-abundance transcripts

  • Implement RNAscope® in situ hybridization for single-molecule detection

  • Use proximity ligation assay (PLA) to detect protein-protein interactions involving NPFFR2

• Quantitative comparative analysis approaches:

  • Standardize detection using calibrated reference standards

  • Implement digital image analysis with machine learning algorithms for IHC quantification

  • Use real-time PCR with appropriate reference genes specifically validated for target tissues

  • Establish tissue microarrays (TMAs) with paired normal/pathological samples for comparative studies

• Experimental design considerations:

  • Include paired normal/tumor samples from the same patient when possible

  • Use larger sample sizes to account for biological variability

  • Implement multiple detection methods for cross-validation

  • Consider single-cell approaches to identify specific cell populations with altered expression

• Validation approaches:

  • Confirm antibody specificity using knockout/knockdown controls

  • Validate results using orthogonal detection methods

  • Perform receptor binding assays with labeled NPFF peptides

  • Use CRISPR-engineered cell lines with endogenous tagging of NPFFR2

Research has shown that 75% of HCC tissues exhibit increased NPFFR2 expression compared to adjacent tissues, making this a valuable model system for protocol optimization .

What is the current understanding of NPFFR2's role in immune regulation and how can researchers effectively study this function?

Emerging research indicates NPFFR2 plays significant roles in immune regulation, particularly in macrophages:

• Current knowledge on NPFFR2 in immune function:

  • NPFFR2 is expressed on various macrophage populations including bone marrow-derived macrophages (BMDMs)

  • NPFF treatment (1 nM) significantly activates NPFFR2 protein expression in macrophages

  • NPFF simultaneously up-regulates and down-regulates large numbers of genes in BMDMs

  • NPFFR2 and NPFF are activated at the spinal cord in rat inflammatory hyperalgesia models

  • NPFF down-regulates nitric oxide levels in RAW 264.7 macrophages and mouse peritoneal macrophages

  • NPFF enhances M2 macrophage activation of adipose tissue macrophages

• Methodological approaches for studying NPFFR2 in immune cells:

  • Transcriptomic analysis: RNA sequencing of immune cells after NPFF treatment

  • Flow cytometry: Detect NPFFR2 expression using appropriate antibodies and compare across immune cell subsets

  • Functional assays: Measure phagocytosis, cytokine production, and nitric oxide production

  • In vivo models: Employ NPFFR2 knockout mice in inflammatory disease models

• Experimental protocols for macrophage studies:

  • BMDMs can be treated with 1 nM NPFF for 18h to study transcriptomic changes

  • NPFFR2 protein expression can be detected by immunofluorescence using antibodies from Novus Biologicals (NPB300-169)

  • For imaging studies, cells should be stained with Hoechst 33342 for nuclei visualization

  • Images can be obtained using confocal microscopy (e.g., LSM510, Carl Zeiss)

This research area represents a significant frontier in understanding NPFFR2 biology beyond its traditional roles in pain modulation and cancer.

How can researchers address non-specific binding issues when using NPFFR2 antibody, HRP conjugated?

Non-specific binding is a common challenge with NPFFR2 antibodies that can be systematically addressed:

• Optimization of blocking conditions:

  • Increase blocking agent concentration (5-10% BSA or serum)

  • Extend blocking time (2-4 hours at room temperature or overnight at 4°C)

  • Include 0.1-0.3% Triton X-100 in blocking buffer for cell permeabilization

  • Use commercial blocking buffers specifically designed to reduce background

• Antibody dilution optimization:

  • Perform titration experiments with serial dilutions (typically 1:100 to 1:10,000)

  • For IHC applications, start with recommended range of 1:20-1:200 and optimize

  • For ELISA applications, determine optimal working dilution experimentally

  • Consider primary antibody incubation at 4°C overnight rather than at room temperature

• Washing protocol refinements:

  • Increase number of washes (5-7 instead of standard 3)

  • Extend washing time (10 minutes per wash)

  • Add 0.05-0.1% Tween-20 to wash buffers

  • Use automated washers for consistent washing efficiency

• Additional measures for reducing background:

  • Pre-absorb antibody with relevant tissue powder

  • Quench endogenous peroxidase with 0.3-3% H₂O₂ treatment before antibody application

  • Use avidin/biotin blocking for tissues with high endogenous biotin

  • Include competitive peptide controls to confirm specificity

For definitive validation, comparing staining patterns between tissues with confirmed high expression (HCC) versus low expression (normal liver) provides valuable confirmation of specificity .

What strategies can resolve inconsistent results when measuring NPFFR2 across different experimental platforms?

Addressing inconsistencies in NPFFR2 detection across platforms requires systematic troubleshooting:

• Sample preparation standardization:

  • Adopt consistent lysis buffers across different assay platforms

  • Standardize protein/RNA extraction protocols

  • Implement consistent storage conditions for all samples

  • Process all compared samples simultaneously when possible

• Platform-specific optimization:

  • For ELISA:

    • Validate antibody pairs for lack of interference

    • Optimize sample dilution to ensure measurements within linear range

    • Include standard curve on each plate for normalization between experiments

  • For IHC/IF:

    • Standardize fixation protocols (duration, temperature)

    • Maintain consistent antigen retrieval conditions (TE buffer pH 9.0 recommended)

    • Use automated staining platforms when possible

    • Implement quantitative image analysis tools

  • For Western blot:

    • Optimize protein loading (10-30 μg typically adequate)

    • Use freshly prepared reagents

    • Ensure consistent transfer conditions

    • Include loading controls for normalization

• Cross-validation approaches:

  • Confirm key findings using orthogonal detection methods

  • Implement spike-recovery experiments to assess matrix effects

  • Compare results with published literature values

  • Verify expression patterns using public databases (e.g., Human Protein Atlas)

• Data normalization strategies:

  • Use reference standards across all experiments

  • Implement batch correction for multi-plate/multi-day experiments

  • Employ appropriate housekeeping genes/proteins validated for stability in your experimental system

  • Consider ratio-based measurements (e.g., NPFFR2/reference protein)

How should researchers approach method validation when studying novel functions of NPFFR2 in previously unexplored cell types?

When investigating NPFFR2 in novel contexts, comprehensive method validation is essential:

• Expression confirmation strategy:

  • Implement multi-modal detection (protein, mRNA, functional assays)

  • Begin with RT-PCR to confirm transcript presence

  • Follow with Western blot using multiple antibodies targeting different epitopes

  • Confirm subcellular localization using immunofluorescence

  • Consider public database mining for preliminary evidence of expression

• Functional validation approaches:

  • Implement CRISPR/Cas9 knockout or siRNA knockdown validation

  • Use selective agonists (NPFF peptides) and antagonists (RF9)

  • Assess canonical downstream signaling (Gi-coupled pathways)

  • Monitor calcium mobilization using fluorescent indicators

  • Evaluate RhoA activation as described in HCC studies

• Controls for novel cell type investigations:

  • Include positive control cells with confirmed NPFFR2 expression (e.g., hepatocellular carcinoma cells)

  • Implement negative controls (siRNA knockdown cells)

  • Use selective antagonist controls

  • Include primary cells from multiple donors to account for individual variations

• Specialized validation for immune cells:

  • Confirm NPFFR2 surface expression via flow cytometry

  • Use PE rat-anti mouse F4/80 antibody and FITC rat anti-mouse CD11b antibody for macrophage identification

  • Consider population heterogeneity in analysis

  • Validate function by observing response to NPFF treatment (1 nM for 18h is a validated condition)

These comprehensive validation approaches establish a solid foundation for novel discoveries while minimizing false positives or artifacts.

What emerging applications might benefit from improved NPFFR2 antibodies?

Several promising research areas could advance with enhanced NPFFR2 antibodies:

• Therapeutic antibody development:

  • Function-blocking antibodies targeting NPFFR2 could provide novel treatments for hepatocellular carcinoma

  • Antibody-drug conjugates delivering cytotoxic agents to NPFFR2-overexpressing cancer cells

  • Bispecific antibodies targeting NPFFR2 and immune checkpoint proteins

  • Nanobody development for improved tissue penetration

• Diagnostic applications:

  • Liquid biopsy development using circulating NPFFR2 detection

  • Multiplexed imaging using NPFFR2 antibodies for cancer subtyping

  • Companion diagnostics to identify patients suitable for NPFFR2-targeted therapies

  • Imaging agents for tumor detection using radiolabeled antibodies

• Pain research applications:

  • Tools to study NPFFR2 dynamics in pain modulation pathways

  • Development of antibodies distinguishing activated vs. inactive receptor states

  • Investigation of NPFFR2 expression changes in chronic pain models

  • Studies of NPFFR2-opioid receptor interactions

• Immune regulation research:

  • Tools to explore NPFFR2's role in various immune cell populations

  • Investigation of receptor expression during inflammation

  • Studies of NPFFR2 in macrophage polarization (M1/M2 balance)

  • Development of antibodies suitable for flow cytometry applications

Recent structural insights from cryo-EM studies provide a foundation for rational design of next-generation antibodies with enhanced specificity and functional properties .

What technical innovations might enhance the specificity and utility of NPFFR2 antibodies?

Emerging technologies offer potential improvements for next-generation NPFFR2 antibodies:

• Structure-guided antibody engineering:

  • Utilize recent cryo-EM structural data to design antibodies targeting conformationally distinct epitopes

  • Develop antibodies that selectively recognize activated receptor states

  • Create antibodies that distinguish between closely related NPFFR1 and NPFFR2 by targeting non-conserved regions

  • Engineer antibodies with enhanced binding to specific domains like ECL2 or N-terminal regions

• Advanced conjugation technologies:

  • Site-specific conjugation to preserve optimal antigen binding

  • Cleavable linkers for improved signal-to-noise ratios

  • Multiplexed labeling strategies (HRP + fluorophore)

  • Novel enzymatic tags for enhanced detection sensitivity

• Affinity maturation approaches:

  • Phage display for selection of higher-affinity variants

  • Yeast display for fine-tuning binding characteristics

  • Directed evolution strategies to enhance specificity

  • Computational design of complementarity-determining regions (CDRs)

• Format innovations:

  • Single-domain antibodies for improved tissue penetration

  • Bispecific antibodies targeting two distinct NPFFR2 epitopes

  • Antibody fragments optimized for specific applications

  • pH-sensitive antibodies for improved internalization

These technological advances could significantly enhance the utility of NPFFR2 antibodies for both research and clinical applications.

How might NPFFR2 research contribute to developing novel pain management strategies?

NPFFR2's involvement in pain modulation pathways suggests several promising research directions:

• Mechanisms of NPFFR2-mediated pain modulation:

  • NPFFR2 functions as a receptor for NPFF neuropeptides which are known as morphine-modulating peptides

  • The receptor mediates its action through G-proteins that activate phosphatidylinositol-calcium second messenger systems

  • Recent structural studies have elucidated the binding modes of RF-amide peptides, providing a foundation for drug development

  • Inhibiting NPFFR2 may provide analgesic effects while potentially reducing opioid-related side effects

• Therapeutic development pathways:

  • Development of bifunctional drugs targeting both opioid receptors and NPFFR2

  • Creation of selective NPFFR2 antagonists based on recent structural insights

  • Investigation of NPFFR2-targeting antibodies as potential analgesics

  • Exploration of small molecule modulators with improved receptor selectivity

• Research approaches to investigate NPFFR2 in pain:

  • Study NPFFR2 expression changes in various pain models

  • Investigate interactions between NPFFR2 and opioid receptors using co-immunoprecipitation

  • Utilize CRISPR/Cas9 knockout models to assess NPFFR2's role in pain perception

  • Implement optogenetic approaches to selectively activate NPFFR2-expressing neurons

• Clinical translation considerations:

  • Biomarker development to identify patients who might benefit from NPFFR2-targeted therapies

  • Development of PET imaging agents to visualize NPFFR2 expression in pain pathways

  • Design of clinical trials specifically targeting conditions with altered NPFFR2 expression

  • Exploration of combination therapies with existing analgesics

Prior multitarget ligands like BN-9 and DN-9 demonstrated efficacy by acting on both opioid receptors and NPFFR2, but lacked precise selectivity between NPFFR2 and NPFFR1 . Enhanced structural understanding now enables more selective drug development.

What is the potential significance of NPFFR2 in immune regulation based on current evidence?

Emerging evidence suggests NPFFR2 plays important roles in immune function:

• Current evidence of NPFFR2 in immune regulation:

  • NPFFR2 is expressed on various macrophage populations, particularly bone marrow-derived macrophages (BMDMs)

  • NPFF treatment (1 nM) significantly activates NPFFR2 protein expression in macrophages

  • RNA sequencing reveals NPFF simultaneously upregulates and downregulates large numbers of genes in BMDMs

  • NPFF enhances M2 macrophage activation of adipose tissue macrophages

  • NPFF downregulates nitric oxide (NO) levels in RAW 264.7 macrophages and mouse peritoneal macrophages

  • NPFF attenuates inflammatory reactions in carrageenan-induced inflammation models

• Potential physiological implications:

  • Modulation of inflammatory responses through macrophage activity regulation

  • Potential role in resolution of inflammation via M2 macrophage polarization

  • Involvement in tissue repair processes through macrophage-mediated mechanisms

  • Possible contribution to metabolic regulation through effects on adipose tissue macrophages

• Research directions to explore immune functions:

  • Characterize NPFFR2 expression across immune cell lineages

  • Investigate receptor dynamics during various inflammatory conditions

  • Study receptor signaling pathways in specific immune cell subsets

  • Develop immune cell-specific knockout models to assess functional significance

• Therapeutic implications:

  • Potential development of NPFFR2-targeting therapies for inflammatory conditions

  • Exploration of receptor modulation for metabolic disorders involving inflammation

  • Investigation of NPFFR2 in tumor-associated macrophages and potential cancer therapies

  • Study of NPFFR2 in context of opioid-induced immune modulation