MCHR2 Antibody

What are the primary applications for MCHR2 antibodies in neuroscience research?

MCHR2 antibodies are valuable tools for investigating several key applications in neuroscience research:

• Protein detection and quantification: Western blot analysis allows detection of MCHR2 in tissue lysates, commonly revealing bands at approximately 38-40 kDa (calculated molecular weight) though observed weight may appear higher (~70-72 kDa) due to post-translational modifications .

• Tissue localization: Immunohistochemistry (IHC) and immunofluorescence (IF) allow visualization of MCHR2 expression patterns in brain regions, particularly in the striatum and hypothalamus .

• Protein-protein interactions: Co-immunoprecipitation experiments can identify MCHR2 binding partners and signaling complexes.

• Receptor activation studies: Antibodies can detect changes in receptor expression or localization following administration of MCH or antagonists .

For optimal experimental design, researchers should consider using multiple detection methods to validate findings, particularly when studying regions with overlapping MCHR1 and MCHR2 expression patterns .

How do I select the appropriate antibody specificity for MCHR2 receptor studies?

Selection of MCHR2 antibodies requires careful consideration of epitope specificity and experimental goals:

• Epitope location considerations:

  • N-terminal domain antibodies (AA 1-39): Useful for detecting full-length receptor but may not recognize truncated forms

  • C-terminal domain antibodies (AA 291-340): Effective for detecting both membrane-bound and internalized receptors

  • Cytoplasmic domain antibodies: Advantageous for detecting intracellular signaling events

• Antibody validation requirements:

  • Confirm specificity using knockout/knockdown controls or peptide blocking experiments

  • Test cross-reactivity, especially when working with models expressing both MCHR1 and MCHR2

  • Verify applications with positive control tissue (human hypothalamic and striatal samples)

For transgenic animal research, consider antibodies raised against the amino acid sequence 237-251 or 283-332, which show reliable detection in experimental models .

What dilutions and protocols are recommended for MCHR2 antibody applications?

Optimal working dilutions and protocols vary by application and specific antibody:

Western Blot:

• Typical dilution range: 1:500-1:2000

• Protein loading: 20-50 μg total protein per lane

• Detection method: Enhanced chemiluminescence systems work well with HRP-conjugated secondary antibodies

Immunohistochemistry:

• Typical dilution range: 1:50-1:300

• Antigen retrieval: Heat-induced epitope retrieval in citrate buffer (pH 6.0)

• Blocking: 5-10% normal serum from secondary antibody species, plus 0.3% Triton X-100

ELISA:

• Typical dilution range: 1:1000-1:20000

• Coating concentration: 1-2 μg/ml of capture antibody

• Detection method: Biotin-conjugated or direct HRP-conjugated antibodies show good sensitivity

For consistent results across experiments, prepare working dilutions fresh and store antibody stocks according to manufacturer recommendations (typically -20°C with minimal freeze-thaw cycles) .

How can I optimize MCHR2 detection in species with differential expression patterns?

MCHR2 expression varies significantly across species, requiring optimized protocols:

• Human and primate tissue:

  • MCHR2 shows robust expression in human hypothalamus and striatum

  • Fresh-frozen tissue yields better results than formalin-fixed paraffin-embedded samples

  • Pre-absorption with MCHR1 peptides can improve specificity in regions with overlapping expression

• Rodent models:

  • Native rodents lack MCHR2, making them inappropriate for MCHR2 antibody validation

  • Transgenic models expressing human MCHR2 (MCHR1R2 mice) require careful antibody selection targeting human-specific epitopes

  • Validate using transgenic controls with confirmed MCHR2 expression profiles

• Other species considerations:

  • Ferrets and dogs express MCHR2 and may serve as alternative models

  • Zebrafish express mchr2 and provide a valuable developmental model system

For cross-species studies, sequence alignment of the antibody epitope against target species is essential before proceeding with experimental applications .

What technical challenges exist in differentiating MCHR1 and MCHR2 immunoreactivity in tissues with co-expression?

Distinguishing between MCHR1 and MCHR2 signals presents several methodological challenges:

• Epitope overlap issues:

  • The two receptors share approximately 37% sequence identity

  • 22 of 33 residues responsible for MCH recognition are conserved between receptors

  • Antibodies targeting conserved regions may cross-react

• Recommended validation strategies:

  • Peptide competition assays with specific blocking peptides for each receptor

  • Sequential immunostaining with MCHR1-specific antibodies followed by MCHR2-specific antibodies

  • Dual immunofluorescence with differentially labeled secondary antibodies

  • Controls using tissues known to express only one receptor type

• Subcellular localization differentiation:

  • MCHR2 may show distinct trafficking patterns due to differential G-protein coupling (Gq vs. Gi/o)

  • High-resolution confocal or super-resolution microscopy can help differentiate receptor populations

RNA-based methods like in situ hybridization with receptor-specific probes can complement antibody-based approaches to confirm specificity of detected signals .

How can I assess functional responses of MCHR2 using antibody-based techniques?

Several antibody-dependent methodologies can assess MCHR2 functional responses:

• Phosphorylation-state specific antibodies:

  • Detect activation-dependent phosphorylation events downstream of Gq-coupling

  • Monitor calcium signaling pathways activated by MCHR2 stimulation

  • Use phospho-specific antibodies against PLC, PKC, or calcium-dependent kinases

• Receptor internalization assays:

  • Surface biotinylation followed by immunoprecipitation with MCHR2 antibodies

  • Antibody feeding assays to track receptor trafficking following MCH stimulation

  • Immunofluorescence with cell-impermeant antibodies to label surface receptors

• Proximity ligation assays:

  • Detect MCHR2 interactions with signaling partners

  • Compare interaction profiles between MCHR1 and MCHR2 in response to ligands

  • Combine with pharmacological interventions to map signaling pathways

When designing these experiments, include appropriate controls to account for non-specific antibody binding and ensure temporal resolution appropriate for the signaling events being studied .

How do MCHR2 antibodies help elucidate ligand binding and receptor activation mechanisms?

MCHR2 antibodies provide critical insights into structure-function relationships:

• Conformational state-specific antibodies:

  • Antibodies targeting the N-terminal domain (AA 1-39) can detect ligand-induced conformational changes

  • Epitopes in ECL2 (extracellular loop 2) regions are particularly sensitive to receptor activation states

  • Conformation-specific antibodies can distinguish between active and inactive receptor states

• Key structural insights revealed through antibody studies:

  • MCH adopts a cysteine-mediated hairpin loop configuration when bound to MCHR2

  • The central arginine from the LGRVY core motif penetrates deeply into the transmembrane pocket

  • ECL2 of MCHR2 forms a hydrophobic cap composed of F177, V181, S183, C184, A185, F186, and L188

• Application in mutagenesis validation:

  • Antibodies detect expression levels of mutant receptors in structure-function studies

  • Help confirm that loss of function in mutants isn't due to impaired expression

  • Support identification of key residues like W2.64 and Y7.32 that introduce steric hindrance affecting MCH binding

For comprehensive structural studies, combining antibody approaches with cryo-EM and computational methods provides the most complete understanding of MCHR2 activation mechanisms .

What considerations are important when using MCHR2 antibodies to study receptor signaling pathways?

Understanding MCHR2 signaling pathways requires specific experimental considerations:

• G-protein coupling detection:

  • MCHR2 exclusively couples to Gq/11 proteins (unlike MCHR1 which couples to Gi/o)

  • Antibodies against activated Gq/11 can confirm receptor-specific signaling

  • Co-immunoprecipitation with MCHR2 antibodies can identify associated G-protein complexes

• Downstream signaling detection:

  • Monitor calcium mobilization and IP3 production as indicators of MCHR2 activation

  • Assess PKC activation and translocation following receptor stimulation

  • Evaluate ERK1/2 phosphorylation as a convergent signaling output

• Experimental design considerations:

  • Include appropriate time points (seconds to minutes) to capture transient signaling events

  • Compare signaling in cells expressing MCHR2 alone versus those co-expressing MCHR1

  • Account for potential heteromerization between MCHR1 and MCHR2 affecting signaling outcomes

Studies have shown that MCHR2 activation can oppose the actions of MCHR1, suggesting complex cross-talk mechanisms that require careful experimental design and interpretation .

What methodological approaches can differentiate between antagonist binding to MCHR1 versus MCHR2?

Distinguishing antagonist selectivity between receptor subtypes requires specialized approaches:

• Antibody-based competitive binding assays:

  • Use labeled antibodies against extracellular epitopes to monitor displacement by antagonists

  • Compare binding profiles between MCHR1 and MCHR2 to identify selective compounds

  • Control for non-specific effects using knockout or knockdown systems

• Structural determinants of selectivity:

  • Position 6.48 contains tryptophan in MCHR1 but alanine in MCHR2, affecting antagonist binding

  • Position 7.42 (G369 in MCHR1) facilitates conformational changes necessary for antagonist binding

  • Position 3.35 differences contribute to binding site architecture differences

• Experimental validation approaches:

  • Site-directed mutagenesis to introduce MCHR1-specific residues into MCHR2

  • Compare antagonist binding profiles between wild-type and mutant receptors

  • Develop chimeric receptors with defined domain swaps to map selectivity determinants

These methodological approaches have revealed that antagonists like SNAP-7941 demonstrate remarkable selectivity for MCHR1 over MCHR2, primarily due to differences in the binding pocket architecture .

How can MCHR2 antibodies be utilized in metabolic disease research models?

MCHR2 antibodies provide valuable tools for investigating metabolic regulation:

• Tissue-specific expression profiling:

  • Hypothalamic expression patterns correlate with feeding behavior regulation

  • Peripheral tissue expression (colon, skeletal muscle, fat) suggests broader metabolic roles

  • Changes in expression levels can be monitored in response to metabolic challenges

• Metabolic phenotype correlation:

  • MCHR1R2 transgenic mice expressing human MCHR2 show resistance to diet-induced obesity

  • These models exhibit lower food intake, improved glucose levels, and improved lipid profiles

  • Antibody-based receptor quantification helps correlate expression levels with metabolic outcomes

• Methodological approach for metabolic studies:

  • Combine immunohistochemistry with metabolic phenotyping

  • Quantify receptor expression changes in response to nutritional interventions

  • Correlate receptor levels with hormonal profiles and body composition measurements

Research has shown that MCHR2 expression can modify MCHR1-mediated effects on metabolism, suggesting complex interplay between these receptor systems that requires careful experimental design and interpretation .

What protocols are effective for detecting MCHR2 in clinical samples for translational research?

Clinical sample analysis requires specialized protocols:

• Tissue preparation considerations:

  • Fresh-frozen human tissues yield optimal results for MCHR2 detection

  • Formalin-fixed samples require extended antigen retrieval (20 minutes at 95°C in citrate buffer)

  • Post-mortem interval significantly affects antibody detection sensitivity

• Recommended applications for clinical samples:

  • Immunohistochemistry (1:50-1:100 dilution) for formalin-fixed tissues

  • Western blot (1:500 dilution) for protein extracts from fresh-frozen samples

  • Quantitative immunofluorescence with standard curves for relative quantification

• Controls and validation for clinical studies:

  • Include known positive control tissues (hypothalamus, striatum)

  • Validate with multiple antibodies targeting different epitopes

  • Consider parallel RNA analysis (qPCR or in situ hybridization) to confirm expression

For longitudinal clinical studies, consistency in sample processing and antibody lots is critical to minimize technical variability that could confound biological interpretations .

How can researchers integrate MCHR2 antibody findings with other molecular profiling techniques?

Multi-modal integration approaches enhance research insights:

• Single-cell analysis integration:

  • Combine MCHR2 immunofluorescence with single-cell RNA sequencing

  • Identify cell type-specific expression patterns and correlate with transcriptional signatures

  • Develop multiplexed immunofluorescence protocols to co-stain with cell-type markers

• Multi-omics integration strategies:

  • Correlate protein expression data from antibody-based studies with transcriptomics

  • Link MCHR2 signaling to metabolomic profiles in response to antagonist treatment

  • Integrate phosphoproteomic data to map signaling networks downstream of MCHR2

• Recommended experimental design:

  • Split samples for parallel processing across different platforms

  • Include common reference samples across experimental batches

  • Implement appropriate normalization strategies to integrate data across modalities

This integrated approach has proven valuable in understanding how MCHR2 signaling networks interact with other molecular systems, particularly in complex physiological processes like energy homeostasis and sleep regulation .

What are common technical issues with MCHR2 antibodies and their solutions?

Researchers frequently encounter specific technical challenges:

• Non-specific binding in Western blots:

  • Issue: Multiple bands appearing at unexpected molecular weights

  • Solution: Increase blocking time (overnight at 4°C) with 5% non-fat dry milk

  • Solution: Include 0.1% SDS in antibody dilution buffer to reduce non-specific interactions

  • Solution: Pre-absorb antibody with non-specific proteins or competitor tissue lysates

• Weak or absent immunohistochemical signal:

  • Issue: Insufficient penetration or epitope masking

  • Solution: Extend antigen retrieval time to 20-30 minutes

  • Solution: Test multiple antibody concentrations (1:50, 1:100, 1:200)

  • Solution: Use tyramide signal amplification for low-abundance detection

• Inconsistent results between experiments:

  • Issue: Antibody degradation or variability between lots

  • Solution: Aliquot antibodies to minimize freeze-thaw cycles

  • Solution: Maintain detailed records of antibody lots and validation results

  • Solution: Include positive control samples in each experimental run

For particularly challenging applications, consider testing multiple antibodies targeting different epitopes to validate findings and optimize detection sensitivity .

How do post-translational modifications affect MCHR2 antibody recognition?

Post-translational modifications significantly impact antibody detection:

• Glycosylation effects:

  • Native MCHR2 shows higher apparent molecular weight (~70-72 kDa) than predicted (38 kDa)

  • N-linked glycosylation sites may mask antibody epitopes

  • Solution: Treatment with PNGase F to remove N-linked glycans can improve detection

  • Epitopes near glycosylation sites may show variable recognition

• Phosphorylation considerations:

  • Agonist-induced phosphorylation alters receptor mobility in SDS-PAGE

  • Phosphorylation at specific residues may create or mask antibody epitopes

  • Solution: Compare detection in samples treated with phosphatase inhibitors versus untreated

• Methodological adjustments:

  • For heavily modified proteins, increase SDS concentration in sample buffer

  • Consider lower percentage gels (7-8%) for better separation of modified receptors

  • Include deglycosylation controls when comparing expression levels between samples

Researchers should be aware that experimental manipulations affecting cellular signaling may alter post-translational modification patterns, potentially affecting antibody recognition independent of expression changes .

What quantitative methods provide reliable MCHR2 protein measurement?

For accurate quantification, several methodological approaches are recommended:

• Western blot quantification:

  • Use total protein normalization rather than single housekeeping proteins

  • Implement standard curves with recombinant protein for absolute quantification

  • Capture images within linear detection range of chemiluminescence

  • Apply consistent analysis protocols across experimental batches

• Quantitative immunohistochemistry/immunofluorescence:

  • Include calibration standards in each staining batch

  • Control for tissue section thickness and antibody penetration depth

  • Use automated image analysis with consistent thresholding parameters

  • Report both intensity and area measurements for comprehensive quantification

• ELISA-based quantification:

  • Develop sandwich ELISA using antibodies targeting different epitopes

  • Include standard curves with recombinant protein fragments

  • Validate across different tissue types to account for matrix effects

  • Implement spike-and-recovery tests to validate assay performance

When reporting quantitative changes, always include both statistical significance and effect size measures, along with detailed methodological parameters to ensure reproducibility .

How can MCHR2 antibodies contribute to drug discovery and development studies?

MCHR2 antibodies play critical roles in drug development workflows:

• Target engagement studies:

  • Competitive binding assays using labeled antibodies to screen potential ligands

  • Conformation-specific antibodies to detect receptor activation states

  • Immunoprecipitation followed by mass spectrometry to identify novel binding partners

• Mechanistic elucidation of drug action:

  • Antibodies detecting receptor internalization following drug exposure

  • Phospho-specific antibodies to map signaling pathway activation

  • Structure-based epitope mapping to correlate with computational drug design models

• Experimental design considerations:

  • Include both acute and chronic drug exposure conditions

  • Assess receptor expression changes in response to antagonist treatment

  • Validate findings across multiple cell lines and primary tissues

Recent structural studies have revealed that antagonists like SNAP-94847 display remarkable selectivity between receptor subtypes due to specific residue differences in the binding pocket, information crucial for developing selective therapeutic agents .

What novel applications of MCHR2 antibodies are emerging in neuroscience research?

Advanced neuroscience applications are expanding the utility of MCHR2 antibodies:

• Circuit-level functional connectivity:

  • Combine retrograde tracing with MCHR2 immunohistochemistry

  • Identify neuronal populations receiving MCH innervation

  • Map receptor distribution across functional circuits regulating sleep and metabolism

• In vivo imaging applications:

  • Develop fluorescently labeled MCHR2 antibody fragments for two-photon microscopy

  • Create activity-dependent labeling strategies using phospho-specific antibodies

  • Incorporate antibody-based sensors for dynamic receptor trafficking visualization

• Therapeutic antibody development:

  • Engineer antibodies targeting specific epitopes to modulate receptor function

  • Develop intrabodies for selective inhibition of intracellular signaling pathways

  • Create bispecific antibodies targeting MCHR2 and components of signaling cascades

Integrating these approaches with genetic tools and physiological measurements provides unprecedented insights into how MCHR2 contributes to complex behaviors and disease states .