QRFPR Antibody

What is QRFPR and why is it significant in research?

QRFPR (Pyroglutamylated RFamide Peptide Receptor), also known as GPR103, is a class A G-protein-coupled receptor that functions as the cognate receptor for the neuropeptide 26RFa (QRFP-26). This receptor system plays crucial roles in energy metabolism, appetite regulation, and various other physiological functions . QRFPR is highly expressed in the brain, particularly in the hypothalamus, trigeminal ganglia, and vestibular neurons, with moderate expression in the amygdala, cortex, pituitary, and other brain regions. In peripheral tissues, it shows expression in the retina, heart, kidney, testis, and thyroid . The receptor's involvement in multiple physiological processes, including potential roles in neurodevelopment through Ca²⁺-dependent PI3K-AKT pathways, makes it a valuable target for neurobiological and metabolic research .

What types of QRFPR antibodies are available for research applications?

Researchers have access to multiple antibodies targeting different epitopes of QRFPR:

Most QRFPR antibodies are rabbit-derived polyclonals that detect endogenous levels of total GPR103/QRFPR protein .

How should I validate QRFPR antibody specificity for my research?

Validating antibody specificity is critical for reliable research outcomes. A comprehensive validation approach includes:

• Blocking peptide controls: Use the immunogenic peptide to pre-absorb the antibody before application. This should abolish specific signals, as demonstrated in western blot analyses with the Anti-QRFPR/GPR103 (extracellular) Antibody (AGR-033) .

• Positive and negative tissue controls: Test antibodies on tissues known to express (brain, hypothalamus) and not express QRFPR. This validation method is employed by manufacturers like Boster Bio .

• Knockout/knockdown validation: When available, use QRFPR knockout or knockdown samples as negative controls.

• Multiple antibody approach: Employ antibodies targeting different epitopes of QRFPR to confirm findings.

• Cross-reactivity assessment: If working with non-human models, verify species cross-reactivity as predicted reactivity may include Pig, Bovine, Horse, Sheep, Rabbit, Dog, and Xenopus for some antibodies .

What are the optimal conditions for Western blot detection of QRFPR?

For optimal Western blot detection of QRFPR:

• Sample preparation: Use membrane fractions for brain tissue or whole cell lysates for cell lines (e.g., LNCaP) . QRFPR is a transmembrane protein, so proper membrane protein extraction protocols are essential.

• Antibody dilution: Typically 1:500 for primary antibodies like Anti-QRFPR/GPR103 (extracellular) Antibody has shown good results in rat and mouse brain membranes and human cell lysates .

• Detection system: HRP-conjugated secondary antibodies with enhanced chemiluminescence provide sensitive detection.

• Expected molecular weight: Be aware that the theoretical molecular weight of QRFPR is approximately 49.5 kDa , but post-translational modifications may alter the apparent molecular weight.

• Controls: Include both positive control tissues (brain membranes) and negative controls (antibody preincubated with blocking peptide) .

How can QRFPR antibodies be utilized in structural and functional studies of the receptor?

Recent advances in QRFPR structural biology provide new opportunities for antibody-based research:

• Epitope mapping: Using antibodies targeting different regions (N-terminal, extracellular loops, internal regions) to understand domain accessibility and conformational changes upon ligand binding.

• Receptor-ligand interactions: Antibodies can be used in co-immunoprecipitation experiments to investigate interactions between QRFPR and its endogenous ligand 26RFa, particularly focusing on the unique assembly mode of the extracellular region of QRFPR with the N-terminus of 26RFa recently identified through cryo-EM studies .

• Conformational studies: Antibodies recognizing specific conformational epitopes can help detect active versus inactive receptor states, important for understanding QRFPR signaling.

• Interaction with signaling partners: Use antibodies in proximity ligation assays or co-immunoprecipitation to study interactions between QRFPR and downstream G-protein partners, particularly given QRFPR's coupling to Gq proteins and regulation of adenylate cyclase activity and intracellular calcium levels .

• Receptor trafficking: Fluorescently-labeled antibodies targeting extracellular epitopes can be used in live-cell imaging to track receptor internalization and recycling.

What considerations are important when studying QRFPR variants using antibodies?

When investigating QRFPR variants, such as the Y68H and R371W variants identified in individuals with intellectual disability :

• Epitope location: Ensure the antibody's epitope does not overlap with the variant site, which could affect binding affinity.

• Variant-specific detection: Consider developing variant-specific antibodies for direct detection of mutant forms.

• Expression level analysis: Use quantitative approaches like Western blotting or ELISA to compare expression levels between wild-type and variant QRFPR.

• Subcellular localization: Employ immunofluorescence to determine if variants show altered trafficking or membrane localization.

• Functional impact assessment: Combine antibody-based detection with functional assays to correlate variant expression with altered signaling outcomes.

• Controls: Include both wild-type and variant constructs in overexpression systems as positive controls when studying endogenous variants.

How should I interpret inconsistent QRFPR antibody staining patterns across different brain regions?

Varied staining patterns across brain regions may reflect biological reality rather than technical issues:

• Expression level differences: QRFPR is differentially expressed across brain regions, with highest levels in the hypothalamus, trigeminal ganglia, and vestibular neurons, and moderate levels in amygdala, cortex, and other regions .

• Receptor isoforms: Consider whether region-specific isoforms might exist that contain different epitopes recognized by your antibody.

• Post-translational modifications: Regional differences in glycosylation or other modifications of QRFPR could affect antibody binding.

• Methodological considerations:

  • Optimize fixation conditions for each region

  • Adjust antibody concentration for regions with different expression levels

  • Consider antigen retrieval methods appropriate for specific brain regions

  • Use multiple antibodies targeting different epitopes to confirm patterns

• Validation approaches: In situ hybridization for QRFPR mRNA can help validate protein expression patterns detected by antibodies.

What are potential causes and solutions for non-specific binding when using QRFPR antibodies?

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

• Causes of non-specific binding:

  • Cross-reactivity with related GPCRs

  • Inadequate blocking

  • Too high antibody concentration

  • Sample overloading

  • Inappropriate fixation causing epitope masking or protein denaturation

• Solutions:

  • Blocking optimization: Test different blocking agents (BSA, normal serum, commercial blockers) and longer blocking times.

  • Antibody dilution series: Establish the optimal concentration through a titration experiment.

  • Wash stringency: Increase number and duration of washes, consider adding low concentrations of detergent.

  • Pre-absorption: Pre-incubate antibody with the immunizing peptide as a specificity control .

  • Alternative antibodies: Try antibodies targeting different epitopes of QRFPR.

  • Sample preparation: Optimize protein extraction and ensure equal loading.

How can QRFPR antibodies contribute to understanding the receptor's role in metabolic disorders?

QRFPR and its ligand 26RFa are implicated in energy metabolism and appetite regulation, offering research opportunities:

• Tissue expression profiling: Use QRFPR antibodies to map expression changes in metabolic tissues (hypothalamus, adipose tissue, pancreas) under different nutritional states or in metabolic disease models.

• Co-localization studies: Combine QRFPR antibodies with markers for specific neuronal populations or with other metabolic regulators to understand circuit integration.

• Receptor regulation: Examine how QRFPR expression and localization change in response to metabolic challenges using quantitative immunohistochemistry or Western blotting.

• Therapeutic targeting validation: Use antibodies to validate the specificity of novel compounds designed to modulate QRFPR activity, such as the 2-aminopyridine antagonist developed to mimic the C-terminal Arg-Phe motif of 26RFa .

• Cross-talk with other systems: Investigate potential interactions between QRFPR and other metabolic regulators through co-immunoprecipitation studies followed by proteomics analysis.

What considerations are important when using QRFPR antibodies in neurodevelopmental research?

Recent findings linking QRFPR variants to intellectual disability highlight its potential role in neurodevelopment :

• Developmental expression profiling: Use antibodies to track QRFPR expression throughout brain development, focusing on critical periods.

• Signaling pathway investigation: Combine QRFPR antibodies with phospho-specific antibodies for downstream effectors in the Ca²⁺-dependent PI3K-AKT pathway implicated in QRFPR's neurodevelopmental roles .

• Cell-type specificity: Determine which neural cell types (neurons, glia) express QRFPR during development using co-immunolabeling.

• Subcellular localization: Track developmental changes in QRFPR subcellular distribution using high-resolution imaging techniques.

• Functional correlation: Correlate QRFPR expression patterns with functional development of specific brain circuits, particularly those involved in cognitive functions affected in intellectual disability.

• Disease model validation: Verify altered QRFPR expression or localization in models of neurodevelopmental disorders using quantitative immunohistochemistry or Western blotting.

What modifications to standard protocols are needed when using QRFPR antibodies for live-cell imaging?

Live-cell imaging of QRFPR requires special considerations:

• Antibody selection: Choose antibodies targeting extracellular domains of QRFPR, such as the N-terminus or extracellular loops. The Anti-QRFPR/GPR103 (extracellular) Antibody targeting amino acid residues 18-32 (N-terminus) would be suitable .

• Antibody fragments: Consider using Fab fragments rather than full IgG to minimize receptor crosslinking and potential activation.

• Fluorophore selection: Choose bright, photostable fluorophores with appropriate spectral properties for your imaging system. Consider pH-sensitive fluorophores for endocytosis studies.

• Labeling strategy: Direct labeling of primary antibodies may be preferable to secondary antibody approaches to minimize background and non-specific binding.

• Physiological conditions: Maintain cells at appropriate temperature and pH during imaging; GPCR trafficking can be sensitive to these parameters.

• Controls: Include controls for antibody specificity (cells not expressing QRFPR) and potential effects of antibody binding on receptor function.

How can QRFPR antibodies be effectively used in studies examining receptor-ligand interactions?

Understanding QRFPR's interaction with its ligand 26RFa can benefit from antibody-based approaches:

• Binding interference assays: Test whether antibodies targeting specific epitopes block 26RFa binding, helping map the ligand-binding domain.

• Conformation-specific detection: Develop or identify antibodies that preferentially recognize the active (ligand-bound) conformation of QRFPR.

• FRET/BRET studies: Combine fluorescently-labeled antibodies with labeled ligand to study binding dynamics through resonance energy transfer techniques.

• Pull-down experiments: Use immobilized antibodies to capture QRFPR-ligand complexes for biochemical analysis.

• Competition assays: Develop assays where antibodies compete with potential novel ligands for receptor binding.