GPR37 Antibody, Biotin conjugated

What is GPR37 and why is it significant in neurological research?

GPR37 (G protein-coupled receptor 37) is an orphan G protein-coupled receptor predominantly expressed in brain regions including the cerebellum, corpus callosum, caudate nucleus, putamen, hippocampus, and substantia nigra. Its significance stems from its role as a substrate of parkin (an E3 ubiquitin ligase) and its association with autosomal recessive juvenile parkinsonism. GPR37 is also known as Pael receptor (Parkin-associated endothelin receptor-like receptor) or ETBR-LP-1 (Endothelin B receptor-like protein 1) . Recent research has revealed its potential function in mediating oligodendrocyte differentiation, though its complete physiological role remains to be fully elucidated, particularly in neurons and other glial cells . The receptor's tendency to become insoluble and unfolded when overexpressed makes it particularly relevant to understanding protein misfolding mechanisms in neurodegenerative conditions .

What are the key specifications of commercially available GPR37 Antibody, Biotin conjugated?

GPR37 Antibody, Biotin conjugated is typically a polyclonal antibody raised in rabbits against a specific peptide sequence (amino acids 114-133) from the human Prosaposin receptor GPR37 protein . These antibodies are purified using antigen affinity methods and presented in liquid form. The formulation generally contains preservatives (0.03% Proclin 300) and stabilizers (50% Glycerol in 0.01M PBS, pH 7.4) . The antibody specifically targets human GPR37 and has been validated for ELISA applications. Commercial preparations require storage at -20°C or -80°C with precautions to avoid repeated freeze-thaw cycles that could compromise antibody integrity .

How does GPR37 relate to Parkinson's disease pathophysiology?

GPR37 has emerged as a potential biomarker for Parkinson's disease (PD) due to several critical observations. In sporadic PD, both GPR37 protein density and mRNA expression are significantly augmented in the substantia nigra . Moreover, the GPR37 ectodomain (ecto-GPR37), which is released from cells by shedding (a phenomenon rarely described for GPCRs), shows increased levels in cerebrospinal fluid (CSF) samples from PD patients compared to neurological controls . This correlation appears to be specific to PD, as similar increases were not observed in Alzheimer's disease patients. Mechanistically, when GPR37 is overexpressed, it tends to become insoluble, unfolded, and ubiquitinated, potentially contributing to dopaminergic neuronal death in juvenile Parkinson disease . The accumulation of this misfolded protein may trigger cellular stress pathways implicated in neurodegenerative processes, providing a direct link between GPR37 dysfunction and parkinsonian pathology.

What are the optimal protocols for using GPR37 Antibody, Biotin conjugated in ELISA experiments?

For ELISA applications using GPR37 Antibody, Biotin conjugated, researchers should implement the following methodological considerations:

• Antibody Dilution: Though specific dilutions may vary between manufacturers, a typical starting dilution ranges from 1:1000 to 1:8000 for immunoassays . Titration experiments are recommended to determine optimal concentrations for specific experimental conditions.

• Sample Preparation: When working with brain tissue, comprehensive homogenization in appropriate lysis buffer containing protease inhibitors is essential. For CSF samples, minimal processing (centrifugation at 1800 × g at 4°C for 10 minutes) followed by immediate storage in aliquots at -80°C is recommended to preserve protein integrity .

• Assay Development for Ecto-GPR37 Detection: For specific quantification of ecto-GPR37 in biological fluids, consider adapting the nanoluciferase-based immunoassay approach described in literature, which has successfully detected GPR37 fragments in CSF samples from PD patients .

• Controls: Include both positive controls (such as brain tissue samples known to express GPR37, particularly from mouse brain, mouse cerebellum, or rat brain tissues) and negative controls to validate assay specificity .

• Detection System: Since the antibody is biotin-conjugated, implement avidin/streptavidin-based detection systems coupled with appropriate enzymes or fluorophores for signal generation and amplification.

• Cross-Reactivity Assessment: Verify the specificity of the antibody against potential cross-reactive proteins, particularly other members of the GPCR family that share structural similarities with GPR37.

What are the critical considerations for storing and handling GPR37 Antibody, Biotin conjugated to maintain its functionality?

Maintaining the integrity and functionality of GPR37 Antibody, Biotin conjugated requires meticulous attention to storage and handling conditions:

• Temperature Management: Store the antibody at -20°C or -80°C immediately upon receipt . For -20°C storage, aliquoting may be unnecessary for smaller volumes (e.g., 20μl sizes containing 0.1% BSA) .

• Freeze-Thaw Cycles: Minimize freeze-thaw cycles as repeated freezing and thawing can significantly compromise antibody activity . Create working aliquots during initial thawing to avoid multiple freeze-thaw cycles.

• Buffer Conditions: The antibody is typically supplied in PBS with 0.02% sodium azide and 50% glycerol at pH 7.3 . Maintaining these buffer conditions is essential for stability.

• Working Dilution Preparation: When preparing working dilutions, use buffers containing protein stabilizers (such as BSA or non-fat milk) to prevent non-specific binding and maintain antibody activity.

• Contamination Prevention: Use sterile techniques when handling the antibody to prevent microbial contamination which can degrade antibody performance.

• Transport Conditions: If transportation between laboratories is necessary, ensure continuous cold chain maintenance using dry ice or specialized shipping containers designed for biological reagents.

• Expiration Monitoring: Document receipt dates and monitor the shelf-life. Most antibody preparations remain stable for approximately one year after shipment when stored properly .

How can researchers validate the specificity of GPR37 Antibody, Biotin conjugated for their experimental systems?

Validating antibody specificity is critical for ensuring reliable experimental results. For GPR37 Antibody, Biotin conjugated, consider implementing these validation strategies:

• Western Blot Analysis: Perform western blotting on tissues known to express GPR37 (e.g., mouse brain, cerebellum, or rat brain) to confirm detection of the expected 67 kDa band .

• Knockout/Knockdown Controls: Utilize samples from GPR37 knockout models or cells treated with GPR37-specific siRNA/shRNA as negative controls to confirm antibody specificity .

• Peptide Competition Assays: Pre-incubate the antibody with excess immunizing peptide (the 114-133AA sequence of human GPR37) before application to samples. Specific antibody binding should be significantly reduced or eliminated.

• Cross-Reactivity Assessment: Test the antibody against samples from multiple species (human, mouse, rat) to confirm expected cross-reactivity patterns as reported in product specifications .

• Immunoprecipitation Followed by Mass Spectrometry: Perform immunoprecipitation using the antibody followed by mass spectrometry analysis to identify captured proteins and confirm specific enrichment of GPR37.

• Parallel Antibody Validation: Compare results with alternative antibodies targeting different epitopes of GPR37, such as anti-human-GPR37-N, anti-mouse-GPR37-N, or anti-pan-GPR37-C antibodies mentioned in the literature .

• Immunohistochemistry Pattern Analysis: Verify that the staining pattern matches the known expression profile of GPR37 in brain tissues (cerebellum, corpus callosum, caudate nucleus, putamen, hippocampus, and substantia nigra).

How can GPR37 Antibody, Biotin conjugated be utilized in studying ecto-GPR37 as a potential biomarker for Parkinson's disease?

Investigating ecto-GPR37 as a PD biomarker requires sophisticated methodological approaches:

• CSF Sample Collection and Processing: Collect CSF samples via lumbar puncture into polypropylene tubes and immediately centrifuge at 1800 × g at 4°C for 10 minutes before aliquoting and storing at -80°C . This protocol minimizes protein degradation and contamination.

• Immunoassay Development: Establish a sensitive detection system similar to the nanoluciferase-based immunoassay reported in literature . This could involve:

  • Capture antibody selection targeting the N-terminal domain of GPR37

  • Using biotin-conjugated GPR37 antibody as the detection antibody

  • Implementing a streptavidin-coupled reporter system for signal generation

  • Optimizing sample dilutions and incubation conditions

• Comparative Cohort Analysis: Design studies including:

  • PD patients (both drug-naïve and treated)

  • Age-matched neurological controls

  • Other neurodegenerative conditions (e.g., Alzheimer's disease) to establish specificity

  • Longitudinal sampling when possible to track biomarker changes over disease progression

• Correlation with Clinical Parameters: Analyze ecto-GPR37 levels in relation to:

  • Disease duration

  • Motor symptom severity (UPDRS scores)

  • Cognitive function measurements

  • Response to therapeutic interventions

  • Other established biomarkers (e.g., α-synuclein levels)

• Mass Spectrometry Validation: Implement liquid chromatography-mass spectrometric analysis to definitively identify specific ecto-GPR37 peptides in CSF samples, providing orthogonal validation of antibody-based detection methods .

• Statistical Analysis Framework: Apply appropriate statistical methods including ROC curve analysis to determine sensitivity and specificity of ecto-GPR37 as a diagnostic or prognostic biomarker for PD.

What experimental approaches can elucidate the relationship between GPR37 shedding mechanisms and Parkinson's disease pathology?

Investigating GPR37 shedding mechanisms requires sophisticated experimental designs:

• Identification of Proteolytic Enzymes: Use protease inhibitor panels and genetic manipulation approaches to identify the specific matrix metalloproteinases (MPs) responsible for GPR37 ectodomain shedding. This is particularly relevant given the altered levels of MPs described in post-mortem brain samples from PD patients .

• Cell Models for Shedding Studies:

  • Establish neuronal cell lines overexpressing GPR37 with epitope or fluorescent tags to monitor shedding

  • Implement primary neuronal cultures from specific brain regions relevant to PD

  • Develop iPSC-derived dopaminergic neurons from PD patients and controls to study shedding in disease-relevant contexts

• In Vivo Shedding Monitoring:

  • Utilize transgenic mouse models expressing tagged versions of GPR37

  • Implement microdialysis techniques to sample brain extracellular fluid for ecto-GPR37 detection

  • Correlate shedding with behavioral phenotypes and neurodegeneration markers

• Mechanistic Studies:

  • Investigate the relationship between α-synuclein accumulation and GPR37 shedding

  • Examine whether parkin dysfunction affects GPR37 processing and shedding

  • Determine if oxidative stress or other PD-relevant cellular stressors modulate GPR37 shedding

• Therapeutic Targeting:

  • Explore whether modulation of GPR37 shedding affects neuronal survival in PD models

  • Test if specific protease inhibitors can normalize GPR37 processing in disease models

  • Investigate potential correlations between therapeutic responses and changes in CSF ecto-GPR37 levels

How can researchers integrate GPR37 antibody-based detection with other techniques to comprehensively study GPR37 biology in neurodegeneration?

A multi-modal approach to GPR37 biology requires integration of various techniques:

• Transcriptomic Analysis: Combine antibody-based protein detection with RT-qPCR for GPR37 mRNA quantification, using appropriate reference genes like β-Glucuronidase for normalization . This approach can reveal discrepancies between transcriptional and translational regulation.

• Multiplex Biomarker Panels:

  • Design panels that simultaneously detect GPR37 and other PD-relevant proteins (α-synuclein, DJ-1, LRRK2)

  • Implement multiplex immunoassays that can analyze multiple analytes from limited biological samples

  • Correlate GPR37 levels with established and emerging biomarkers

• Imaging Approaches:

  • Apply proximity ligation assays to study GPR37 interactions with potential binding partners

  • Implement super-resolution microscopy to examine GPR37 localization in cellular compartments

  • Use intravital imaging in animal models to track GPR37 expression and processing in real-time

• Functional Studies:

  • Couple antibody detection with electrophysiological recordings to correlate GPR37 levels with neuronal activity

  • Implement calcium imaging to examine if GPR37 expression or shedding affects neuronal signaling

  • Design reporter assays to monitor downstream signaling pathways potentially regulated by GPR37

• Single-Cell Analysis:

  • Combine immunostaining with single-cell RNA sequencing to identify cell populations with unique GPR37 expression profiles

  • Implement spatial transcriptomics to map GPR37 expression patterns in complex brain tissues

  • Correlate single-cell GPR37 protein levels with cellular phenotypes and vulnerability to degeneration

What are common technical issues encountered when using GPR37 Antibody, Biotin conjugated and how can they be resolved?

Several technical challenges may arise when working with GPR37 Antibody, Biotin conjugated:

• High Background Signal:

  • Cause: Insufficient blocking, excessive antibody concentration, or non-specific binding

  • Solution: Optimize blocking conditions (try different blocking agents such as BSA, non-fat milk, or commercial blockers); titrate antibody dilutions (starting from 1:1000-1:8000) ; include additional washing steps with detergent-containing buffers

• Weak or No Signal Detection:

  • Cause: Insufficient protein expression, antibody degradation, or incompatible detection system

  • Solution: Verify GPR37 expression in samples (use positive controls like mouse brain tissue) ; ensure proper antibody storage conditions; confirm compatibility between biotin conjugation and detection system; consider signal amplification methods

• Multiple Bands in Western Blots:

  • Cause: Protein degradation, post-translational modifications, or non-specific binding

  • Solution: Include protease inhibitors during sample preparation; verify expected molecular weight (67 kDa) ; perform peptide competition assays to confirm specificity

• Batch-to-Batch Variability:

  • Cause: Differences in antibody production or purification

  • Solution: Maintain reference samples for comparative analysis between batches; consider purchasing larger quantities of a single lot for long-term studies

• Sample Matrix Interference:

  • Cause: Components in biological samples interfering with antibody binding

  • Solution: Optimize sample dilution; test different buffer formulations; implement pre-clearing steps to remove interfering components

• Inconsistent ELISA Results:

  • Cause: Temperature fluctuations, pipetting errors, or plate variability

  • Solution: Standardize laboratory conditions; use calibrated pipettes; implement technical replicates; consider automated liquid handling systems for high-throughput applications

How should researchers validate experimental results when studying ecto-GPR37 as a potential biomarker for Parkinson's disease?

Rigorous validation is essential when evaluating ecto-GPR37 as a PD biomarker:

• Cohort Selection and Characterization:

  • Ensure well-defined patient and control groups with detailed clinical characterization

  • Include both drug-naïve and treated PD patients to account for potential medication effects

  • Match groups for age, gender, and other demographic factors that might influence biomarker levels

• Sample Quality Assessment:

  • Implement standardized protocols for CSF collection and processing (polypropylene tubes, immediate centrifugation at 1800 × g at 4°C)

  • Establish quality control metrics (e.g., cell counts, protein concentration, hemoglobin content) to identify compromised samples

  • Document pre-analytical variables (time from collection to processing, storage duration)

• Analytical Validation:

  • Determine assay precision through intra- and inter-assay coefficient of variation calculations

  • Establish limits of detection and quantification for the specific ecto-GPR37 assay

  • Perform spike-and-recovery experiments to assess matrix effects in CSF samples

• Orthogonal Method Confirmation:

  • Validate antibody-based detection with mass spectrometry identification of specific ecto-GPR37 peptides

  • Consider alternative antibodies targeting different epitopes to confirm findings

  • Correlate protein measurements with mRNA expression data where appropriate

• Statistical Rigor:

  • Implement appropriate statistical methods with correction for multiple comparisons

  • Perform power calculations to ensure adequate sample sizes

  • Consider machine learning approaches for multivariate analysis when combining ecto-GPR37 with other biomarkers

• Longitudinal Validation:

  • Design follow-up studies to track biomarker changes over disease progression

  • Correlate biomarker levels with clinical outcomes and disease milestones

  • Assess potential effects of therapeutic interventions on biomarker levels

What are emerging applications of GPR37 Antibody, Biotin conjugated in neurodegenerative disease research beyond Parkinson's disease?

While GPR37 has been primarily studied in the context of Parkinson's disease, several emerging research directions warrant exploration:

• Other Synucleinopathies:

  • Investigate GPR37 expression and ecto-GPR37 levels in multiple system atrophy and dementia with Lewy bodies

  • Examine whether different α-synuclein pathologies distinctly affect GPR37 processing and function

  • Determine if GPR37 could serve as a differential biomarker across the spectrum of synucleinopathies

• Oligodendrocyte-Related Pathologies:

  • Given GPR37's role in oligodendrocyte differentiation , explore its involvement in demyelinating disorders

  • Investigate potential connections to multiple sclerosis pathophysiology

  • Examine GPR37 in models of oligodendrocyte injury and regeneration

• Protein Misfolding Disorders:

  • Study GPR37's propensity to become insoluble and ubiquitinated as a model for understanding protein quality control mechanisms

  • Explore potential interactions with other aggregation-prone proteins beyond α-synuclein

  • Investigate whether GPR37 misfolding contributes to ER stress in various neurodegenerative contexts

• Developmental Neurobiology:

  • Examine GPR37's expression during neural development and potential roles in neurogenesis

  • Investigate whether developmental GPR37 expression patterns predispose specific neuronal populations to later degeneration

  • Study potential roles in adult neurogenesis and neural repair mechanisms

• Neuroinflammatory Processes:

  • Explore potential interactions between GPR37 and neuroinflammatory signaling pathways

  • Investigate whether GPR37 shedding is altered during neuroinflammation

  • Examine potential roles in glial-neuronal communication in disease contexts

How might GPR37 antibody-based research contribute to therapeutic development for Parkinson's disease?

GPR37 antibody-based research offers several pathways toward therapeutic development:

• Biomarker-Guided Clinical Trials:

  • Use ecto-GPR37 levels to stratify patients for clinical trials, potentially identifying subgroups more likely to respond to specific interventions

  • Implement ecto-GPR37 monitoring as a pharmacodynamic marker to assess therapeutic efficacy

  • Develop companion diagnostics for emerging PD therapeutics based on GPR37 biology

• Target Validation Strategies:

  • Utilize antibodies to validate GPR37 as a direct therapeutic target

  • Develop function-modulating antibodies that could stabilize GPR37 conformation or prevent pathological interactions

  • Investigate whether preventing GPR37 accumulation or misfolding offers neuroprotective benefits

• Drug Discovery Platforms:

  • Implement cell-based assays using GPR37 antibodies to screen compound libraries for molecules that normalize GPR37 processing

  • Develop high-throughput screening approaches to identify modulators of GPR37 shedding

  • Design assays to discover compounds that prevent the toxic accumulation of misfolded GPR37

• Gene Therapy Approaches:

  • Use antibody-based research to validate potential benefits of GPR37 silencing or overexpression in specific neural populations

  • Develop targeted delivery strategies for GPR37-modulating gene therapies

  • Design reporter systems using GPR37 antibodies to monitor the efficacy of gene therapy approaches in real-time

• Immunotherapy Possibilities:

  • Explore whether antibodies targeting specific GPR37 epitopes could have therapeutic benefits by modulating receptor function or clearing pathological forms

  • Investigate if removing excess ecto-GPR37 from cerebrospinal fluid could alter disease progression

  • Develop antibody engineering approaches to enhance blood-brain barrier penetration for potential GPR37-targeted immunotherapies