GPR55 Antibody

What is GPR55 and where is it expressed in the nervous system?

GPR55 is a G protein-coupled receptor initially identified as a novel cannabinoid receptor. Quantitative RT-PCR studies have revealed GPR55 gene expression across multiple regions of the mouse brain, including the frontal cortex, striatum, hippocampus, and cerebellum . The receptor is a 36.6 kilodalton protein in humans, and orthologs have been identified in various species including canine, porcine, monkey, mouse and rat models .

GPR55 expression has been detected using various methodologies including in situ hybridization (ISH), immunohistochemistry (IHC), and fluorescent ligand binding assays, though expression levels are typically low in neural tissues . While GPR55 mRNA and immunostaining signals have been observed in neurons in the striatum and hippocampus, the specific neuronal phenotypes expressing GPR55 are still being characterized.

What types of GPR55 antibodies are currently available for research?

Multiple types of GPR55 antibodies are currently available from commercial suppliers, with varying applications and specifications:

• Polyclonal antibodies: These represent the majority of available antibodies and are raised against various epitopes, including C-terminus regions of human GPR55 .

• Application-specific antibodies: Suppliers offer antibodies optimized for specific applications including Western Blot (WB), Enzyme-Linked Immunosorbent Assay (ELISA), Immunohistochemistry (IHC), Immunocytochemistry (ICC), Immunofluorescence (IF), and Flow Cytometry (FCM) .

• Species-reactive antibodies: Most commercially available antibodies recognize human GPR55, though some cross-react with other species including rat, mouse, and canine models .

It's worth noting that human GPR55 shows approximately 75% and 78% homology with rat and mouse GPR55 proteins respectively, which can affect antibody recognition across species .

What are the fundamental considerations when selecting a GPR55 antibody?

When selecting a GPR55 antibody for research, consider these critical factors:

• Validation status: Prioritize antibodies with documented validation studies, particularly those tested in GPR55 knockout models or with competing peptides.

• Application compatibility: Ensure the antibody has been validated for your specific application (WB, IHC, ELISA, etc.).

• Target species homology: Consider the sequence homology between your experimental model and the immunogen used to generate the antibody.

• Epitope location: Antibodies targeting different regions of GPR55 may yield different results.

• Detection sensitivity: GPR55 is often expressed at low levels in neural tissues, necessitating antibodies with high sensitivity.

Research has shown that commercially available antibodies against human or bovine GPR55 may not be optimal for detecting GPR55 receptor proteins in mice, despite sequence homology, highlighting the importance of species-specific validation .

How should GPR55 antibody specificity be validated?

Validating GPR55 antibody specificity requires a multi-faceted approach:

• Genetic validation: Compare immunostaining or Western blot results between wild-type and GPR55 knockout models. Studies have reported that some commercial antibodies produce immunostaining signals in GPR55-KO mice, suggesting potential non-specific binding .

• Peptide competition assays: Pre-absorb the antibody with the immunizing peptide before application. Research has shown that GPR55-immunostaining can sometimes still be detected in the presence of the antibody immune peptides, indicating possible cross-reactivity .

• Alternative detection methods: Complement antibody-based approaches with nucleic acid detection methods like RNAscope in situ hybridization to correlate protein and mRNA expression patterns .

• Fluorescent ligand binding: Consider using fluorescent GPR55 ligands like Tocrifluor T1117 to verify receptor localization, particularly in studies using CB1-KO mice to exclude potential CB1 receptor binding .

• Multiple antibody comparison: Use antibodies from different suppliers or those targeting different epitopes to corroborate findings.

What are the optimal protocols for using GPR55 antibodies in brain tissue immunohistochemistry?

When performing immunohistochemistry with GPR55 antibodies on brain tissue, consider these methodological recommendations:

• Fixation optimization: Test different fixation protocols, as overfixation may mask epitopes while underfixation could compromise tissue integrity.

• Antigen retrieval: Implement antigen retrieval steps (heat or enzymatic) to unmask epitopes that might be cross-linked during fixation.

• Signal amplification: Consider using tyramide signal amplification or other sensitive detection systems given the typically low expression levels of GPR55.

• Co-localization studies: Employ double-labeling techniques with established neuronal markers like TH (tyrosine hydroxylase) to characterize GPR55-expressing cell populations. Studies have shown that GPR55-like signals detected in the ventral tegmental area (VTA) were not co-localized with TH-immunostaining in dopaminergic neurons .

• Appropriate controls: Always include both positive controls (tissues known to express GPR55) and negative controls (either GPR55 knockout tissues or primary antibody omission).

• Cross-validation: Complement IHC findings with other methodologies such as RNAscope in situ hybridization or fluorescent ligand binding assays .

How can GPR55 signaling be functionally assessed in neuronal systems?

Assessing GPR55 signaling functionality in neuronal systems involves several complementary approaches:

• Electrophysiological methods: Patch-clamp recordings can detect changes in synaptic transmission following application of GPR55 agonists like O-1602. Studies have shown that GPR55 activation modulates the frequency and magnitude of miniature excitatory postsynaptic currents within the dorsal raphe nucleus .

• Calcium imaging: GPR55 activation can trigger intracellular calcium mobilization, which can be measured using calcium-sensitive dyes or genetically encoded calcium indicators.

• Phosphorylation assays: Measuring phosphorylation of downstream targets, such as NMDA and AMPA receptors, can indicate GPR55 activation. Research has demonstrated that GPR55-mediated cognitive enhancement is underpinned by the phosphorylation of these receptors .

• Neurotransmitter release measurements: In vivo microdialysis can assess how GPR55 activation affects neurotransmitter release, such as dopamine or glutamate in specific brain regions .

• Behavioral paradigms: Functional outcomes of GPR55 signaling can be assessed through behavioral tests examining learning and memory, such as novel object recognition tests (NORT), Y-maze, and Morris water maze (MWM) .

What are the major challenges in detecting GPR55 protein in brain tissue samples?

Researchers face several significant challenges when detecting GPR55 in brain tissue:

• Low expression levels: GPR55 is typically expressed at low levels in the brain, often near the detection threshold of conventional methods. Studies have noted that detected GPR55 signals in the striatum and hippocampus are "very weak" .

• Antibody specificity concerns: Multiple studies have reported issues with antibody specificity, with some commercial antibodies producing signals in GPR55 knockout tissues. Both Abcam and Cayman GPR55 antibodies have shown immunostaining in GPR55-KO mice, suggesting non-specific binding .

• Cellular heterogeneity: GPR55 may be expressed in specific neuronal subpopulations or in non-neuronal cells, requiring single-cell resolution techniques.

• Post-translational modifications: Potential modifications might affect antibody recognition or protein extraction efficiency.

• Receptor internalization: As with many GPCRs, GPR55 may undergo internalization upon activation, affecting its detection at the cell membrane.

To address these challenges, researchers should employ multiple detection methods, rigorous controls, and consider advanced techniques like RNAscope, single-cell RNA sequencing, or proximity ligation assays.

How do I interpret contradictory results when using different GPR55 antibodies?

When facing contradictory results from different GPR55 antibodies, follow this systematic approach:

• Compare antibody characteristics: Examine differences in host species, immunogen sequence, epitope location, and production method (monoclonal vs. polyclonal).

• Review validation data: Assess how each antibody was validated—knockout models, peptide competition, and correlation with mRNA expression provide stronger evidence of specificity.

• Consider epitope accessibility: Different experimental conditions may affect epitope accessibility. Antibodies targeting different regions of GPR55 may perform differently depending on fixation, tissue processing, or protein denaturation methods.

• Evaluate cellular context: GPR55 may form heteromers with other receptors or undergo conformational changes in different cell types, potentially affecting antibody recognition.

• Implement orthogonal validation: Use non-antibody-based methods (genetic reporters, ligand binding, functional assays) to validate contradictory antibody results. Research has employed fluorescent ligand binding assays with Tocrifluor T1117 as an alternative approach to assess GPR55 expression .

• Consider publication bias: Published studies demonstrating successful GPR55 detection may overshadow negative results or specificity concerns.

How can GPR55 antibodies be used to investigate its role in synaptic plasticity and cognitive functions?

GPR55 antibodies can be strategically deployed to investigate its role in synaptic plasticity and cognition:

• Subcellular localization studies: High-resolution imaging techniques like super-resolution microscopy combined with GPR55 antibodies can determine the receptor's distribution at synapses. This localization information provides insight into potential functional roles.

• Activity-dependent regulation: GPR55 antibodies can be used to assess whether receptor expression or distribution changes following learning tasks or induction of synaptic plasticity.

• Co-immunoprecipitation assays: GPR55 antibodies can identify protein interaction partners involved in synaptic function, potentially revealing mechanistic insights.

• Correlative structure-function studies: Combining GPR55 immunolabeling with electrophysiological recordings or calcium imaging can correlate receptor expression with functional outcomes.

• Targeted manipulation approaches: GPR55 antibody-based approaches (like receptor internalization induced by antibody binding) combined with behavioral assessment can reveal causal relationships.

Research has demonstrated that GPR55 activation through agonists like O-1602 can ameliorate learning and memory deficits in mouse models, potentially through mechanisms involving serotonin synthesis and synaptic transmission in the dorsal raphe nucleus .

What methodological approaches can be used to study GPR55 effects on neurotransmitter systems?

Investigating GPR55 effects on neurotransmitter systems requires sophisticated methodological approaches:

• In vivo microdialysis: This technique can measure changes in extracellular levels of neurotransmitters like dopamine, glutamate, or serotonin following administration of GPR55 agonists like O-1602 .

• Electrophysiological recordings: Patch-clamp studies can assess how GPR55 activation modulates synaptic transmission. Research has shown that GPR55 agonists can alter miniature excitatory postsynaptic currents in the dorsal raphe nucleus .

• Genetic knockdown approaches: Targeted knockdown of key enzymes (like tryptophan hydroxylase 2 for serotonin synthesis) can determine whether GPR55 effects are mediated through specific neurotransmitter systems. Studies have shown that TPH2 knockdown in the dorsal raphe nucleus reduces the beneficial effects of GPR55 activation on learning and memory .

• Neurotransmitter synthesis assays: GPR55 antibodies combined with antibodies against key synthetic enzymes (like TPH2 for serotonin) can reveal co-localization and potential regulatory relationships. Research has demonstrated that GPR55 activation enhances TPH2 expression and promotes serotonin synthesis .

• Behavioral pharmacology: Combining GPR55 agonists with neurotransmitter-specific antagonists can determine which neurotransmitter systems mediate specific behavioral effects.

How can I optimize Western blot protocols for GPR55 detection?

Optimizing Western blot protocols for GPR55 detection requires careful attention to several technical aspects:

• Sample preparation:

  • Use fresh tissue or cells when possible

  • Consider membrane-enriched fractions since GPR55 is a membrane protein

  • Include protease inhibitors to prevent degradation

  • Test different lysis buffers (RIPA vs. milder detergents) as harsh detergents may disrupt epitope structure

• Protein denaturation:

  • Test different denaturation conditions (temperature, reducing agents)

  • For membrane proteins like GPR55, avoid extended boiling which can cause aggregation

• Gel selection and transfer:

  • Use gradient gels (10-15%) for better resolution around 36.6 kDa (reported mass of GPR55)

  • Consider wet transfer methods for better transfer efficiency of membrane proteins

  • Use PVDF membranes which typically perform better for hydrophobic proteins

• Blocking and antibody incubation:

  • Test multiple blocking agents (milk vs. BSA) as milk proteins can sometimes bind non-specifically to hydrophobic regions

  • Optimize primary antibody concentration and incubation time (typically start with 1:500-1:1000 dilution)

  • Consider overnight incubation at 4°C to improve signal-to-noise ratio

• Detection system:

  • Consider enhanced chemiluminescence or fluorescence-based systems for improved sensitivity

  • Include appropriate positive controls (transfected cells overexpressing GPR55)

  • Always include molecular weight markers to confirm band identity

• Validation controls:

  • If possible, include samples from GPR55 knockout models as negative controls

  • Consider peptide competition assays if knockout samples are unavailable

What strategies can improve reproducibility when working with GPR55 antibodies?

Improving reproducibility with GPR55 antibodies requires systematic approaches:

• Standardized protocols:

  • Develop detailed protocols specifying all parameters (buffer compositions, incubation times, temperatures)

  • Maintain consistent lot numbers of antibodies when possible, or validate new lots against previous ones

  • Document all experimental conditions, including sample preparation methods

• Quality control measures:

  • Regularly test antibody performance using positive and negative controls

  • Consider creating internal reference standards (e.g., brain tissue lysates with confirmed GPR55 expression)

  • Implement intra- and inter-assay controls to assess variability

• Multi-method validation:

  • Confirm key findings using alternative detection methods

  • Combine antibody-based approaches with mRNA detection techniques

  • Consider functional assays to complement expression studies

• Data transparency:

  • Keep detailed records of all antibody characteristics (catalog number, lot, dilution)

  • Document image acquisition parameters and analysis methods

  • Consider pre-registering experimental protocols for critical studies

• Internal standards:

  • Use loading controls appropriate for your experimental context

  • Consider multiplexing with established markers for specific cell types

  • Include calibration standards when quantifying protein levels

How can GPR55 antibodies contribute to understanding neurological disorders?

GPR55 antibodies can facilitate research into neurological disorders through these approaches:

• Expression profiling: GPR55 antibodies can map receptor distribution in healthy versus diseased tissues. Research has already identified GPR55 expression in the frontal cortex, striatum, hippocampus, and cerebellum—regions implicated in various neurological conditions .

• Biomarker development: Changes in GPR55 expression patterns could potentially serve as biomarkers for disease progression or treatment response.

• Mechanistic studies: GPR55 antibodies can help elucidate the receptor's role in pathological processes. For instance, research has shown that GPR55 activation ameliorates learning and memory deficits in maternal separation models, suggesting potential neuroprotective functions .

• Target validation: GPR55 antibodies can confirm the presence of the receptor in specific cell populations before testing therapeutic modulators.

• Drug development support: Antibodies can help characterize the effects of potential therapeutic compounds on GPR55 expression, localization, or downstream signaling.

• Circuit-specific analysis: Combined with neuronal markers, GPR55 antibodies can identify specific circuits affected in neurological disorders, particularly in regions like the dorsal raphe nucleus where GPR55 activation has been shown to modulate serotonergic transmission and cognitive function .