GPR55 Antibody, Biotin conjugated

What is GPR55 and why is it important in research?

GPR55 is a G-protein-coupled receptor encoded by the GPR55 gene (Gene ID: 9290, Swiss Prot: Q9Y2T6). It functions as a receptor for L-alpha-lysophosphatidylinositol (LPI), which induces Ca²⁺ release from intracellular stores via the heterotrimeric G protein GNA13 and RHOA. GPR55 may be involved in hyperalgesia associated with inflammatory and neuropathic pain. It also plays a significant role in bone physiology by regulating osteoclast number and function, and in neurodevelopment by modulating axon growth and target innervation . As a putative cannabinoid receptor, GPR55 is relevant to studies investigating endocannabinoid signaling systems and potential therapeutic applications.

What is a biotin-conjugated GPR55 antibody?

A biotin-conjugated GPR55 antibody is an immunoglobulin that specifically recognizes and binds to the GPR55 protein and has been chemically linked to biotin molecules. This biotin conjugation enhances the antibody's utility in various detection systems due to the strong affinity between biotin and streptavidin/avidin. Commonly available GPR55 biotin-conjugated antibodies are polyclonal, derived from rabbits immunized with synthetic peptides corresponding to specific amino acid sequences of human GPR55 (such as regions 141-240/319 or 203-222) . This conjugation allows for amplified signal detection and versatility in experimental applications.

What are the primary applications for GPR55 biotin-conjugated antibodies?

GPR55 biotin-conjugated antibodies are primarily used in:

• Western Blot (WB): To detect and quantify GPR55 protein expression in tissue or cell lysates, typically at dilutions between 1:300-1:5000 .

• ELISA (Enzyme-Linked Immunosorbent Assay): For quantitative determination of GPR55 in solution, typically at dilutions between 1:500-1:1000 .

• Immunohistochemistry (IHC): To visualize GPR55 expression in tissue sections when used with appropriate detection systems.

• Immunofluorescence: For cellular localization studies, particularly when investigating GPR55's subcellular distribution (primarily in cell membranes) .

The biotin conjugation provides amplification advantages through secondary detection with streptavidin-conjugated reporter molecules.

What tissue/cell types are known to express GPR55?

GPR55 has been detected in various tissues and cell types:

• Central nervous system: Particularly in the retina during development where it regulates growth cone morphology and axon growth .

• Bone marrow: Where it may influence osteoclast function and bone remodeling .

• Immune cells: Contributing to inflammatory responses.

• Endocrine tissues: With potential roles in metabolic regulation.

• Cancer cells: With emerging evidence for involvement in cancer cell proliferation and migration.

When designing experiments with GPR55 antibodies, researchers should consider that expression levels may vary significantly between tissues, developmental stages, and pathological conditions.

How do GPR55 agonists and antagonists affect antibody binding and experimental outcomes?

The presence of GPR55 ligands in experimental systems can significantly influence antibody binding characteristics and experimental outcomes. When GPR55 binds agonists like lysophosphatidylinositol (LPI) or O-1602, the receptor undergoes conformational changes that can potentially mask or expose epitopes recognized by antibodies . Antagonists like cannabidiol (CBD) or ML192 derivatives may stabilize the receptor in an inactive conformation.

In immunodetection experiments:

• Epitope accessibility may be altered depending on receptor activation state

• Pre-treatment of samples with ligands might require adjusted antibody concentrations

• Fixation methods can differentially preserve ligand-induced conformations

For functional studies, researchers should consider whether antibody binding itself might have agonistic or antagonistic effects on receptor function. Control experiments comparing antibody binding in the presence and absence of known ligands can help interpret complex results, especially in live cell applications.

What are the implications of GPR55 knockout models for antibody validation?

GPR55 knockout (gpr55-/-) models provide crucial controls for antibody validation and specificity determination. Research shows that neurons from gpr55-/- mouse embryos exhibit smaller growth cones, fewer growth cone filopodia, and decreased axonal outgrowth compared to wild-type neurons . These models offer several advantages for antibody validation:

• True negative controls: Tissue from knockout animals should show no specific staining, allowing identification of non-specific binding.

• Validation of developmental phenotypes: GPR55-/- mice show decreased branching in the dorsal terminal nucleus and lower levels of eye-specific segregation of retinal projections in the superior colliculus and dorsal lateral geniculate nucleus .

• Ligand response verification: In absence of GPR55, pharmacological effects of GPR55 ligands (LPI, O-1602, CBD) are not observed, confirming antibody specificity .

When validating biotin-conjugated GPR55 antibodies, comparing staining patterns between wild-type and knockout tissues across multiple detection methods (WB, IHC, IF) provides comprehensive evidence of specificity and appropriate experimental conditions.

How do post-translational modifications of GPR55 affect antibody recognition?

GPR55, like other GPCRs, undergoes various post-translational modifications (PTMs) that can affect antibody recognition. Although specific data on GPR55 PTMs is limited in the provided search results, general principles apply:

• Phosphorylation: Occurs during receptor desensitization and may alter epitope accessibility, particularly for antibodies targeting intracellular domains.

• Glycosylation: GPR55 contains potential N-glycosylation sites in extracellular domains that might affect antibody binding to these regions.

• Palmitoylation: May influence receptor trafficking and membrane localization, potentially affecting antibody accessibility in intact cells.

• Ubiquitination: Can signal receptor degradation and may affect detection in protein turnover studies.

When designing experiments, researchers should consider:

• Using multiple antibodies targeting different epitopes to ensure comprehensive detection

• Comparing native versus denatured conditions to assess conformational epitope recognition

• Employing enzymatic treatments (phosphatases, glycosidases) to evaluate PTM effects on detection

• Validating results across different cell types where PTM patterns may vary

What are the optimal protocols for using biotin-conjugated GPR55 antibodies in Western blotting?

For optimal Western blot results with biotin-conjugated GPR55 antibodies, researchers should consider the following protocol recommendations:

Sample Preparation:

• Extract proteins using buffers containing protease inhibitors to prevent GPR55 degradation

• For membrane proteins like GPR55, include gentle detergents (0.5-1% Triton X-100 or CHAPS)

• Heat samples at 37°C instead of boiling to prevent aggregation of membrane proteins

Electrophoresis and Transfer:

• Use 10-12% polyacrylamide gels for optimal resolution of GPR55 (~37 kDa)

• Transfer to PVDF membranes (preferred over nitrocellulose for hydrophobic proteins)

• Verify transfer efficiency with reversible staining

Immunodetection:

• Block with 3-5% BSA in TBS with 0.1% Tween-20 (BSA is preferred over milk for biotin-conjugated antibodies)

• Dilute primary antibody 1:300-1:5000 in blocking buffer

• Incubate overnight at 4°C for highest sensitivity

• Use streptavidin-HRP (1:2000-1:10000) for detection

• Develop using enhanced chemiluminescence

Controls:

• Include GPR55-overexpressing positive control

• Use GPR55 knockout or knockdown samples as negative controls

• Consider peptide competition assays to confirm specificity

This approach maximizes detection sensitivity while minimizing background and non-specific binding commonly encountered with membrane proteins like GPR55.

How can biotin-conjugated GPR55 antibodies be utilized in colocalization studies?

Biotin-conjugated GPR55 antibodies are valuable tools for colocalization studies examining GPR55's spatial relationship with other proteins. The following methodology optimizes their use:

Sample Preparation:

• Fix cells/tissues with 4% paraformaldehyde (10-15 minutes)

• Permeabilize with 0.1-0.3% Triton X-100 for intracellular epitopes

• For membrane preservation, consider milder detergents like 0.1% saponin

Staining Protocol:

• Block with 5% normal serum and 1% BSA

• Apply biotin-conjugated GPR55 antibody (typically 2-5 μg/ml)

• For colocalization, simultaneously or sequentially apply unconjugated primary antibodies against proteins of interest

• Detect GPR55 using streptavidin conjugated to a fluorophore spectrally distinct from secondary antibodies used for other targets

• Include DAPI for nuclear counterstaining

Imaging Considerations:

• Use confocal microscopy to minimize out-of-focus fluorescence

• Capture sequential scans to prevent crosstalk between channels

• Apply appropriate controls for spectral bleed-through

• Consider super-resolution techniques for detailed subcellular localization

Quantitative Analysis:

• Calculate Pearson's or Mander's coefficients for quantitative colocalization assessment

• Perform line scan analysis across cellular regions to confirm spatial relationships

• Consider pixel-by-pixel intensity correlation analysis

This approach enables precise determination of GPR55's spatial relationship with potential interacting partners, signaling components, or cellular compartments.

What is the optimal approach for using GPR55 biotin-conjugated antibodies in flow cytometry?

For optimal flow cytometry results with biotin-conjugated GPR55 antibodies, implement the following protocol:

Cell Preparation:

• Harvest cells using enzyme-free dissociation methods to preserve membrane proteins

• Maintain viability above 95% for reliable results

• Adjust to 1×10^6 cells per sample

Surface Staining Protocol:

• Block with 2% FBS in PBS for 15 minutes

• Apply biotin-conjugated GPR55 antibody (1-5 μg/ml) for 30 minutes at 4°C

• Wash twice with cold PBS containing 2% FBS

• Incubate with streptavidin-fluorophore conjugate (e.g., streptavidin-PE, APC, or Alexa Fluor dyes)

Intracellular Staining Protocol (if needed):

• Fix cells with 2-4% paraformaldehyde for 10 minutes

• Permeabilize with 0.1% saponin or commercially available permeabilization buffers

• Proceed with blocking and staining as above

Controls:

• Include unstained cells for autofluorescence assessment

• Use isotype-biotin controls followed by streptavidin-fluorophore

• Include secondary-only controls (streptavidin-fluorophore only)

• Use cells with known GPR55 expression levels as positive controls

• When possible, include GPR55 knockout or knockdown cells as negative controls

Analysis Considerations:

• Gate on viable, single cells

• Compare signal to appropriate negative controls

• Consider compensation if using multiple fluorophores

This approach enables quantitative assessment of GPR55 expression across cell populations, facilitating studies of expression dynamics under various conditions or treatments.

How can researchers address non-specific binding issues with biotin-conjugated GPR55 antibodies?

Non-specific binding is a common challenge with biotin-conjugated antibodies. For GPR55 detection, consider these strategies:

Sources of Non-specific Binding:

• Endogenous biotin in samples (particularly in brain, liver, and kidney tissues)

• Cross-reactivity with structurally similar GPCRs

• Fc receptor interactions in immune cells

• Hydrophobic interactions with membrane proteins

Optimization Strategies:

• Biotin Blocking:

  • Pretreat samples with avidin/biotin blocking kits

  • Include free biotin (10-100 μg/ml) in blocking buffers

• Blocking Optimization:

  • Use 3-5% BSA instead of milk (which contains endogenous biotin)

  • Add 0.1-0.3% Triton X-100 to reduce hydrophobic interactions

  • Include 5-10% serum from the same species as secondary reagents

• Antibody Dilution:

  • Perform careful titration experiments to determine optimal concentration

  • For GPR55 biotin-conjugated antibodies, start with manufacturer recommendations (1:300-1:5000 for WB, 1:500-1000 for ELISA)

• Stringency Adjustment:

  • Increase salt concentration (150-500 mM NaCl) in wash buffers

  • Add 0.05-0.1% SDS to wash buffers for Western blotting

  • Increase number and duration of washes

• Validation Controls:

  • Use peptide competition assays with the immunizing peptide

  • Include GPR55 knockout or knockdown samples

  • Compare multiple antibodies targeting different GPR55 epitopes

By systematically implementing these strategies, researchers can significantly reduce non-specific binding while maintaining sensitivity for specific GPR55 detection.

What are the considerations for detecting GPR55 in different species using biotin-conjugated antibodies?

When extending GPR55 detection across species, researchers must carefully consider antibody specificity and epitope conservation:

Species Reactivity Analysis:

• Available biotin-conjugated GPR55 antibodies show confirmed reactivity with human GPR55

• Predicted reactivity extends to mouse, rat, dog, horse, chicken, and rabbit for some antibodies

• Epitope sequence conservation should be verified by sequence alignment

Cross-Species Validation Approach:

• Sequence Comparison:

  • Align the immunogen sequence (e.g., human GPR55 amino acids 141-240 or 203-222) with target species

  • Calculate percent identity and similarity in the epitope region

  • Identify potential species-specific post-translational modifications

• Positive Controls:

  • Use tissues with known GPR55 expression from the target species

  • Consider transfection controls with species-specific GPR55 constructs

  • Compare detection patterns between human and target species samples

• Knockout Validation:

  • When available, use tissues from GPR55 knockout animals of the target species

  • Compare staining patterns between wild-type and knockout samples

• Optimization for Each Species:

  • Adjust antibody concentration (generally higher for less conserved epitopes)

  • Modify fixation protocols based on tissue characteristics

  • Adapt blocking conditions to minimize species-specific background

• Detection System Considerations:

  • Ensure secondary detection reagents (streptavidin conjugates) lack cross-reactivity with target species proteins

  • Consider endogenous biotin levels which vary between species and tissues

By following this systematic approach, researchers can confidently extend GPR55 studies across species while maintaining experimental rigor and reproducibility.

How do different fixation and permeabilization methods affect GPR55 antibody detection?

Fixation and permeabilization protocols significantly impact GPR55 detection due to its membrane localization and conformation-sensitive epitopes:

Fixation Effects:

• Paraformaldehyde (PFA) Fixation:

  • 2-4% PFA generally preserves GPR55 epitopes while maintaining cellular architecture

  • Shorter fixation times (10-15 minutes) minimize epitope masking

  • Preferred for immunofluorescence and flow cytometry applications

• Methanol Fixation:

  • Can improve detection of some intracellular epitopes

  • May disrupt membrane structure and alter conformational epitopes

  • Test side-by-side with PFA for specific antibody clones

• Glutaraldehyde:

  • Provides superior ultrastructural preservation

  • Often causes excessive cross-linking that masks epitopes

  • If needed, use at low concentrations (0.1-0.5%) in combination with PFA

Permeabilization Considerations:

• For Transmembrane and Extracellular Epitopes:

  • Gentle permeabilization or no permeabilization is preferred

  • Digitonin (0.001-0.01%) selectively permeabilizes plasma membrane

  • Saponin (0.1-0.3%) creates small pores while preserving membrane structure

• For Intracellular Epitopes:

  • Triton X-100 (0.1-0.3%) provides reliable access to intracellular domains

  • Consider fixation-permeabilization sequence (simultaneous vs. sequential)

Optimization Strategy:

• Test matrix of conditions combining:

  • Fixation agent and concentration

  • Fixation duration and temperature

  • Permeabilization method and timing

  • Antigen retrieval options (heat, pH, enzymatic)

• Evaluate results based on:

  • Signal intensity at expected molecular weight/location

  • Background levels and non-specific binding

  • Morphological preservation of subcellular structures

These considerations are particularly important when studying GPR55 conformation changes induced by ligands like LPI, O-1602, or CBD, as described in neurodevelopmental studies .

How can biotin-conjugated GPR55 antibodies be used to study GPR55 agonist and antagonist mechanisms?

Biotin-conjugated GPR55 antibodies provide valuable tools for investigating ligand interactions and receptor responses:

Receptor Internalization Assays:

• Track GPR55 redistribution following agonist treatment (LPI, O-1602)

• Compare surface vs. internal receptor pools using non-permeabilized and permeabilized conditions

• Quantify internalization kinetics through time-course experiments

Conformational Change Detection:

• Some antibodies may preferentially recognize active or inactive receptor conformations

• Compare antibody binding before and after treatment with agonists (LPI) versus antagonists (CBD, ML192 derivatives)

• Use proximity ligation assays to detect changes in protein-protein interactions following ligand binding

Ligand Screening Applications:

• Develop competition binding assays using biotin-conjugated antibodies

• Measure displacement of antibody binding by potential ligands

• Compare results with functional assays (β-arrestin recruitment, calcium mobilization)

Data Analysis Considerations:

• Account for different efficacies of ligands (LPI vs. O-1602)

• Consider biased signaling where ligands preferentially activate different pathways

• Compare data with results from thienopyrimidine derivatives and other GPR55 antagonists

This approach enables detailed mechanistic studies of GPR55 pharmacology, potentially identifying novel ligands with therapeutic applications in inflammatory pain, cancer, or neurodevelopmental disorders.

What insights can GPR55 antibody studies provide about its role in neurodevelopment?

GPR55 antibody-based studies offer critical insights into neurodevelopmental processes as evidenced by research on retinal ganglion cell projections:

Developmental Expression Patterns:

• Biotin-conjugated GPR55 antibodies can map temporal and spatial expression throughout neurodevelopment

• Compare expression between wild-type and GPR55 knockout models to correlate with phenotypic differences

• Analyze co-expression with developmental markers and axon guidance molecules

Growth Cone Dynamics:

• Studies show GPR55 regulates growth cone morphology and filopodia formation

• Antibody labeling can visualize receptor distribution within growth cone structures

• Live imaging with minimally disruptive labeling techniques can track receptor dynamics during axon extension

Target Innervation Analysis:

• GPR55 knockout mice show decreased branching in the dorsal terminal nucleus (DTN)

• Antibody labeling can quantify receptor levels at key decision points during innervation

• Combined with anterograde tracing, can correlate receptor expression with branching patterns

Mechanistic Insights:

• Antibody blocking experiments can complement pharmacological approaches

• Phospho-specific antibodies (if available) can track activation of downstream signaling pathways

• Co-immunoprecipitation with biotin-conjugated antibodies can identify developmental binding partners

These approaches have revealed that GPR55 plays important roles in eye-specific segregation of retinal projections in the superior colliculus and dorsal lateral geniculate nucleus, suggesting broader implications for activity-dependent refinement of neural circuits .

How can GPR55 biotin-conjugated antibodies be used in multiplex detection systems?

Biotin conjugation makes GPR55 antibodies particularly suitable for multiplex detection strategies:

Sequential Multiplex Immunohistochemistry:

• Use biotin-conjugated GPR55 antibodies as one component in multiplexed tissue staining

• Apply tyramide signal amplification for signal enhancement and subsequent antibody stripping

• Cycle through multiple rounds of staining to detect 5-10 proteins on the same section

• Useful for characterizing GPR55-expressing cell populations in complex tissues

Mass Cytometry (CyTOF) Applications:

• Conjugate GPR55 antibodies to metal isotopes via biotin-streptavidin bridges

• Integrate into panels of 30+ markers for deep cellular phenotyping

• Quantify GPR55 expression across diverse cell populations simultaneously

Proximity-Based Detection:

• Pair biotin-conjugated GPR55 antibodies with antibodies against potential interacting proteins

• Apply proximity ligation assay (PLA) to visualize interactions as fluorescent puncta

• Quantify interaction dynamics under different ligand conditions (LPI, CBD)

Bead-Based Multiplex Assays:

• Couple streptavidin-coated beads with biotin-conjugated GPR55 antibodies

• Develop pull-down assays to capture GPR55 and associated proteins

• Analyze using flow cytometry or imaging cytometry platforms

Spatial Transcriptomics Integration:

• Combine antibody detection with in situ hybridization techniques

• Correlate protein expression with mRNA localization

• Map receptor distribution across tissue sections with transcriptional profiles

This multiplexing capability enables comprehensive characterization of GPR55 in complex biological systems, providing insights into its diverse functions across different cellular contexts and disease states.