GPR37 Antibody

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

GPR37 (G protein-coupled receptor 37) is an orphan G protein-coupled receptor that plays significant roles in neuronal function. It is also known by several alternative names including EDNRBL, PAELR, hET(B)R-LP, prosaposin receptor GPR37, and ETBR-LP-1 . The protein has a molecular mass of approximately 67.1 kilodaltons and is predominantly expressed in brain tissue .

GPR37's significance in neuroscience stems from its involvement in several critical physiological pathways. It functions as a receptor for multiple ligands including prosaposin, osteocalcin, and neuroprotectin D1, with ligand binding typically inducing endocytosis followed by ERK phosphorylation cascades . Additionally, GPR37 plays important roles in oligodendrocyte differentiation, central nervous system myelination, and resolving inflammatory pain, making it a target of interest in neurological and neurodegenerative disease research .

What are the common applications for GPR37 antibodies in research?

GPR37 antibodies are utilized across multiple experimental applications in neuroscience and molecular biology research. The most common applications include:

These applications enable researchers to detect, localize, and quantify GPR37 expression in various experimental contexts . When selecting a GPR37 antibody, researchers should consider the specific application requirements, as different antibodies may be optimized for particular techniques or experimental conditions .

What species reactivity is available for GPR37 antibodies?

GPR37 antibodies are available with reactivity to multiple species, which is an important consideration when designing experiments. Based on the available data, commonly available reactivity profiles include:

• Human (Hu): Most commercial antibodies target human GPR37

• Rat (Rt): Many antibodies cross-react with rat GPR37

• Mouse (Ms): Several antibodies recognize mouse GPR37

• Predicted reactivity for additional species such as rabbit and other mammals

When selecting an antibody, it's advisable to verify the specific species reactivity through product documentation or validation data, especially when working with less common model organisms. For instance, the polyclonal antibody described in search result reacts with human and rat GPR37, with predicted reactivity to mouse and rabbit, making it versatile for comparative studies across these species .

What are the recommended protocols for GPR37 immunohistochemistry?

Successful immunohistochemical detection of GPR37 requires specific protocol optimizations. The following methodology is recommended based on established research practices:

• Tissue preparation: Standard fixation with paraformaldehyde followed by paraffin embedding or frozen sectioning.

• Antigen retrieval: This step is critical for GPR37 detection. Boil tissue sections for 20 minutes in 10 mM sodium citrate buffer (pH 6.0), followed by cooling to room temperature .

• Blocking: Incubate sections for 2 hours in blocking solution containing 1% normal goat serum, 5% bovine serum albumin, 0.2% fish gelatin, 0.1% Triton X-100, and 0.1% NaN₃ in PBS .

• Primary antibody incubation: Apply anti-GPR37 antibody at a concentration of 1 μg/mL and incubate overnight at 4°C . Typical dilution ranges are 1:50-1:500 depending on the specific antibody .

• Secondary antibody and detection: Use biotinylated anti-rabbit IgG followed by peroxidase-conjugated streptavidin and diaminobenzidine (DAB) color development for chromogenic detection . For fluorescent detection, use appropriate fluorophore-conjugated secondary antibodies.

• Controls: Include negative controls (omitting primary antibody) and positive controls (known GPR37-expressing tissues such as brain) to validate specificity.

This protocol can be adapted for specific experimental needs, but the antigen retrieval step is particularly important for successful GPR37 detection .

How can I optimize Western blot conditions for detecting GPR37?

Optimizing Western blot conditions for GPR37 detection requires attention to several key parameters:

• Sample preparation:

  • For brain tissue samples, use RIPA buffer supplemented with protease inhibitors

  • Include phosphatase inhibitors if phosphorylation status is relevant

  • Complete homogenization and adequate solubilization are critical for membrane proteins like GPR37

• Gel electrophoresis:

  • Use 8-10% SDS-PAGE gels to properly resolve the 67.1 kDa GPR37 protein

  • Load adequate protein amounts (typically 20-50 μg of total protein per lane)

• Transfer conditions:

  • For this membrane protein, use PVDF membrane rather than nitrocellulose

  • Consider longer transfer times (90-120 minutes) or semi-dry transfer systems

• Antibody concentrations:

  • Primary antibody: Begin with 1:500-1:1000 dilution and optimize as needed

  • Secondary antibody: Typically 1:5000-1:10000 dilution

• Detection system:

  • Enhanced chemiluminescence (ECL) systems provide good sensitivity

  • Longer exposure times may be needed depending on expression levels

• Controls:

  • Include brain tissue as a positive control

  • Consider using GPR37 knockout samples as negative controls for specificity validation

By methodically optimizing these parameters, researchers can improve the specificity and sensitivity of GPR37 detection in Western blot applications.

What challenges might I encounter when working with GPR37 antibodies?

Working with GPR37 antibodies presents several challenges that researchers should anticipate:

• Poor plasma membrane expression: GPR37 exhibits poor plasma membrane expression when expressed in most cell types, which can complicate detection in heterologous expression systems . This may lead to inconsistent results when comparing different cell types.

• Potential cross-reactivity: Due to sequence homology with related receptors, particularly GPR37L1, antibodies may exhibit cross-reactivity. Thorough validation using knockout controls is recommended to confirm specificity .

• Antigen retrieval requirements: Unlike many proteins, GPR37 detection in fixed tissues requires stringent antigen retrieval procedures (boiling in citrate buffer), which may damage some tissue structures or epitopes .

• Variable expression levels: GPR37 expression varies significantly across brain regions, potentially resulting in detection challenges in regions with lower expression.

• Post-translational modifications: GPR37 undergoes various post-translational modifications that may mask epitopes or alter antibody recognition depending on cellular context.

To address these challenges, researchers should thoroughly validate antibodies in their specific experimental systems, include appropriate controls, and consider complementary detection methods to confirm findings.

How can I enhance surface expression of GPR37 for functional studies?

Enhancing surface expression of GPR37 is critical for functional studies, as this receptor exhibits poor plasma membrane localization in most expression systems. Three complementary approaches have been established to improve GPR37 trafficking to the cell surface:

• N-terminal truncation:

  • Removing the first 210 amino acids of the N-terminus significantly enhances plasma membrane insertion

  • Complete N-terminal truncation provides maximal surface expression enhancement

  • This approach is particularly useful for structure-function studies that do not require the N-terminal domain

• Co-expression with partner receptors:

  • Co-expressing GPR37 with adenosine receptor A2AR or dopamine receptor D2R significantly increases GPR37 surface expression

  • Co-immunoprecipitation experiments confirm that GPR37 (particularly truncated forms) can robustly associate with D2R

  • This approach may alter ligand binding properties of the partner receptor, as D2R shows modified affinity for both agonists and antagonists when co-expressed with GPR37

• Co-expression with PDZ scaffolds:

  • Syntenin-1, a PDZ scaffold protein, specifically interacts with GPR37

  • This interaction dramatically enhances GPR37 surface expression in HEK-293 cells

  • The approach leverages natural cellular trafficking machinery to improve membrane insertion

These methods can be used individually or in combination depending on the specific experimental requirements and the functional aspects of GPR37 being investigated.

What is known about GPR37's role in neurological disease models?

GPR37's involvement in neurological disease processes has been investigated through various disease models with several key findings:

• Parkinson's disease connections:

  • GPR37 is known as the parkin-associated endothelin receptor-like receptor (PAELR)

  • Accumulation of unfolded GPR37 can trigger ER stress and contribute to neuronal death

  • In parkin-deficient models, GPR37 accumulation has been associated with pathological outcomes

• Demyelinating conditions:

  • GPR37 plays a negative regulatory role in oligodendrocyte differentiation and myelination during development through activation of the ERK1/2 signaling pathway

  • This suggests GPR37 may influence the stability of myelin or resistance to demyelination

  • GPR37 antibodies have been used to study these processes in models of multiple sclerosis and other demyelinating disorders

• Inflammatory pain models:

  • Upon activation by neuroprotectin D1 (NPD1), GPR37 promotes phagocytosis in macrophages

  • It shifts cytokine release toward an anti-inflammatory profile

  • These mechanisms help reverse inflammatory pain in relevant models

  • Extracellular vesicles containing prosaposin bind to macrophage GPR37 to increase expression of the efferocytosis receptor TIM4, accelerating resolution of inflammation

• Sepsis protection:

  • The increased macrophage phagocytosis mediated by GPR37 may provide protection against sepsis during pathogen infection

These diverse roles make GPR37 antibodies valuable tools for investigating multiple neuropathological conditions and potential therapeutic approaches.

How does GPR37 differ from GPR37L1 in experimental detection and function?

While GPR37 and GPR37L1 are related receptors, they exhibit important differences in their cellular distribution, detection methods, and functional roles that researchers should consider:

Understanding these differences is critical when designing experiments to investigate specific functions of GPR37 versus GPR37L1, and when interpreting results of antibody-based detection methods.

What controls should be included when using GPR37 antibodies?

Proper experimental controls are essential for reliable interpretation of results when using GPR37 antibodies. The following controls should be included:

• Negative controls:

  • Omission of primary antibody while maintaining all other protocol steps

  • Isotype controls using non-specific IgG at the same concentration as the primary antibody

  • When available, GPR37 knockout or knockdown samples provide the most stringent negative control

  • Testing tissues known to lack GPR37 expression (non-neural tissues can serve this purpose)

• Positive controls:

  • Mouse or rat brain tissue samples, which are known to express GPR37

  • Heterologous expression systems (e.g., HEK-293 cells) transfected with GPR37 expression constructs

  • For enhanced detection, consider using N-terminally truncated GPR37 constructs which show improved surface expression

• Specificity controls:

  • Peptide competition assays to confirm epitope specificity

  • Comparison of multiple antibodies targeting different epitopes of GPR37

  • Western blot analysis paralleling immunohistochemistry to confirm appropriate molecular weight detection

• Technical controls:

  • Standardized positive samples used across experiments to control for batch effects

  • Internal loading or staining controls appropriate to the experimental technique

Implementing these controls systematically will significantly enhance data reliability and facilitate accurate interpretation of GPR37 antibody-based experiments.

How can I differentiate between GPR37 and its related receptors?

Differentiating between GPR37 and related receptors, particularly GPR37L1, requires careful experimental design and validation:

• Antibody selection strategy:

  • Choose antibodies raised against unique, non-conserved regions of GPR37

  • Avoid antibodies targeting regions with high sequence homology to GPR37L1 or other G protein-coupled receptors

  • Validate antibody specificity through Western blot analysis comparing tissues with different expression patterns of GPR37 and GPR37L1

• Cellular co-localization approach:

  • Utilize the distinct cellular distribution patterns: GPR37 is predominantly neuronal, while GPR37L1 is primarily expressed in microglia and astrocytes

  • Perform double-labeling immunofluorescence using cell-type specific markers:

    • NeuN or MAP2 for neurons (expected to co-localize with GPR37)

    • GFAP for astrocytes (expected to co-localize with GPR37L1)

    • Iba1 for microglia (expected to show strong co-localization with GPR37L1)

• Functional validation:

  • Leverage the differential responses to ligands: GPR37 and GPR37L1 may show distinct signaling patterns in response to prosaposin or other ligands

  • Use receptor-specific knockdown or knockout approaches to confirm antibody specificity

• Differential expression analysis:

  • Compare expression patterns following specific stimuli or in disease models, as GPR37 and GPR37L1 respond differently to neurological injury

By combining these approaches, researchers can achieve reliable differentiation between GPR37 and related receptors, ensuring accurate interpretation of experimental results.

What are the recommended fixation and antigen retrieval methods for GPR37 immunodetection?

Optimal fixation and antigen retrieval are critical for successful GPR37 immunodetection. Based on established protocols, the following methods are recommended:

• Fixation recommendations:

  • For tissue sections: 4% paraformaldehyde fixation for 24-48 hours at 4°C provides good preservation of GPR37 epitopes

  • For cultured cells: 4% paraformaldehyde for 15-20 minutes at room temperature

  • Avoid over-fixation, which can mask GPR37 epitopes, particularly in membrane regions

• Antigen retrieval requirements:

  • Heat-induced epitope retrieval is essential for GPR37 detection in fixed tissues

  • Recommended protocol: Boil sections for 20 minutes in 10 mM sodium citrate buffer (pH 6.0)

  • Allow sections to cool in the same buffer before proceeding with immunostaining

  • This step is critical and cannot be omitted for GPR37 detection, unlike some other proteins

• Alternative retrieval methods:

  • For difficult samples, consider using Tris-EDTA buffer (pH 9.0) as an alternative retrieval solution

  • Some researchers report improved results with pressure cooker-based antigen retrieval methods

• Buffer considerations:

  • pH is critical: while citrate buffer (pH 6.0) works well, some antibodies may perform better with slightly higher pH buffers

  • Addition of 0.05% Tween-20 to retrieval buffers can improve penetration

• Sample-specific optimizations:

  • Frozen sections may require shorter retrieval times (10-15 minutes)

  • Older fixed samples often benefit from extended retrieval periods

These methodological details are particularly important as improper fixation or inadequate antigen retrieval are among the most common causes of false-negative results when detecting GPR37 in tissue samples .

How is GPR37 research contributing to understanding neurodegenerative diseases?

GPR37 research is providing valuable insights into neurodegenerative disease mechanisms through several avenues:

• Parkinson's disease connections:

  • As a parkin-associated receptor (PAELR), GPR37 accumulation in the absence of functional parkin may contribute to neurodegeneration

  • Studies using GPR37 antibodies have revealed abnormal receptor aggregation in dopaminergic neurons in Parkinson's disease models

  • The relationship between GPR37 misfolding and endoplasmic reticulum stress represents a potential therapeutic target

• Myelination and demyelinating disorders:

  • GPR37's negative regulatory role in oligodendrocyte differentiation and myelination via ERK1/2 signaling suggests its involvement in demyelinating conditions

  • Researchers are investigating whether modulating GPR37 activity could enhance remyelination in multiple sclerosis and other demyelinating disorders

  • GPR37 antibodies are essential tools for monitoring these processes in disease models

• Neuroinflammatory mechanisms:

  • GPR37 activation by neuroprotectin D1 promotes anti-inflammatory responses in macrophages

  • This activity may have neuroprotective effects in conditions with neuroinflammatory components

  • The receptor's role in promoting efferocytosis (clearance of apoptotic cells) via TIM4 upregulation suggests potential relevance to neurodegenerative diseases where clearance mechanisms are impaired

• Wnt/β-catenin signaling interactions:

  • Emerging evidence suggests GPR37 may protect LRP6 from ER-associated degradation, thereby promoting Wnt/β-catenin signaling

  • This pathway is implicated in several neurodegenerative conditions and represents a potential mechanism through which GPR37 influences neuronal survival

These research directions highlight the importance of high-quality GPR37 antibodies in elucidating disease mechanisms and potentially identifying novel therapeutic approaches.

What are the emerging applications of GPR37 antibodies in neuroscience research?

GPR37 antibodies are finding novel applications beyond traditional protein detection, opening new avenues in neuroscience research:

• Receptor trafficking studies:

  • Antibodies targeting extracellular epitopes of GPR37 are being used in live-cell imaging to track receptor internalization dynamics

  • This approach is revealing how ligands like prosaposin and neuroprotectin D1 regulate receptor localization and signaling

  • These methods are helping to elucidate the unusual trafficking challenges faced by GPR37

• Proximity-based interaction mapping:

  • Combining GPR37 antibodies with proximity ligation assays allows visualization of protein-protein interactions in situ

  • This technique has revealed previously unknown interactions between GPR37 and other neuronal proteins

  • The approach is particularly valuable for studying associations with D2 dopamine receptors and adenosine A2A receptors

• Single-cell expression profiling:

  • Antibody-based flow cytometry and single-cell Western blot techniques are enabling researchers to characterize GPR37 expression in heterogeneous neural populations

  • These approaches are revealing cell type-specific expression patterns that were previously obscured in whole-tissue analyses

• In vivo functional modulation:

  • Function-blocking antibodies against GPR37 extracellular domains are being developed as tools to modulate receptor activity in vivo

  • This approach offers advantages over genetic knockout models by allowing temporal control of receptor inhibition

• Biomarker development:

  • Studies are exploring whether GPR37 levels in cerebrospinal fluid or exosomes, detected using sensitive antibody-based assays, could serve as biomarkers for neurodegenerative conditions

These emerging applications highlight the continued importance of developing and characterizing high-quality GPR37 antibodies for advancing neuroscience research.

What key considerations should guide my selection of GPR37 antibodies?

When selecting GPR37 antibodies for research applications, several key considerations should guide your decision-making process:

• Experimental application compatibility:

  • Different applications (WB, IHC, IF, etc.) may require antibodies with distinct properties

  • For Western blotting, prioritize antibodies validated for denaturing conditions

  • For immunohistochemistry, select antibodies compatible with your fixation method and requiring appropriate antigen retrieval protocols

• Species reactivity requirements:

  • Confirm that the antibody recognizes GPR37 from your species of interest

  • Cross-reactivity between human, mouse, and rat is common but should be verified

  • Consider whether predicted reactivity is sufficient or if demonstrated reactivity is necessary

• Epitope location considerations:

  • Antibodies targeting the N-terminal region may show limited detection in cells with poor surface expression

  • For trafficking studies, consider using antibodies targeting extracellular domains

  • For detection regardless of trafficking state, antibodies targeting intracellular loops or C-terminus may be preferable

• Validation evidence:

  • Prioritize antibodies with published validation data in applications similar to yours

  • Look for validation using knockout/knockdown controls

  • Multiple antibodies against different epitopes providing consistent results offer stronger evidence

• Technical specifications:

  • Consider format (unconjugated vs. conjugated), host species, and clonality based on your experimental design

  • Review recommended dilutions and incubation conditions for your specific application

By carefully evaluating these factors, researchers can select the most appropriate GPR37 antibodies for their specific experimental needs, maximizing the likelihood of obtaining reliable and interpretable results.

How can I contribute to improving GPR37 antibody validation standards?

Researchers can contribute to improving GPR37 antibody validation standards through several practical approaches:

• Implement comprehensive validation protocols:

  • Test antibodies across multiple applications and experimental conditions

  • Include positive and negative controls, particularly GPR37 knockout or knockdown samples

  • Document batch-to-batch variation by retaining reference samples

• Contribute to public knowledge:

  • Publish detailed methods sections that include complete antibody information (catalog numbers, lots, dilutions, incubation conditions)

  • Share validation data even when results are negative

  • Submit antibody validation data to community resources and antibody validation databases

• Apply multiple detection methods:

  • Confirm antibody results using complementary techniques (e.g., mRNA expression, reporter systems)

  • Use orthogonal approaches such as mass spectrometry to validate Western blot results

  • Employ multiple antibodies targeting different epitopes to strengthen confidence in findings

• Develop improved tools:

  • Generate and characterize new antibodies against underrepresented epitopes

  • Develop tagged GPR37 constructs that can serve as validation tools

  • Consider creating inducible expression systems for controlled validation experiments

• Collaborative standardization:

  • Participate in multi-laboratory validation studies

  • Contribute to the development of standard operating procedures for GPR37 detection

  • Engage with antibody manufacturers to improve product validation information