ADGRG3 Antibody, HRP conjugated

What is ADGRG3 and why are antibodies against it important?

ADGRG3 (Adhesion G Protein-Coupled Receptor G3), also known as GPR97, is a member of the adhesion GPCR family characterized by an extended extracellular region with N-terminal protein modules linked to a TM7 region via a GPCR-Autoproteolysis INducing (GAIN) domain . This receptor has been identified as playing important roles in inflammatory processes, particularly in neutrophils where it forms part of a macromolecular complex with CD177, PAR2, and CD16b . The structural components of GPR97 include a small cysteine-stabilized helical N-terminal domain (NTD) followed by an unusually small GAIN domain consisting of subdomain A with one α-helix and subdomain B with 13 β-strands .

Antibodies against ADGRG3/GPR97 are crucial research tools that enable detection, localization, and functional analysis of this receptor in various experimental contexts. They facilitate studies into the receptor's roles in inflammatory responses, signaling pathways, and potential involvement in disease processes. The specificity of these antibodies for different epitopes or domains of ADGRG3/GPR97 allows researchers to investigate distinct aspects of the receptor's structure and function .

What are the key applications for ADGRG3 Antibody, HRP conjugated?

The ADGRG3 Antibody, HRP conjugated (such as product code CSB-PA769807LB01HU) is primarily designed for enzyme-linked immunosorbent assay (ELISA) applications . The horseradish peroxidase (HRP) conjugation provides direct enzymatic detection capability without requiring secondary antibodies, which streamlines experimental workflows and potentially increases sensitivity.

Key applications include:

• ELISA-based detection and quantification of ADGRG3/GPR97 in cell or tissue lysates

• Protein-protein interaction studies, particularly when investigating ADGRG3/GPR97's binding partners

• Studies involving the macromolecular complex formed by ADGRG3/GPR97 with other proteins such as CD177, mPR3, PAR2, and CD16b in inflammatory processes

• Validation of expression in different cell types or tissues

While the primary validated application is ELISA, researchers should determine optimal working dilutions for their specific experimental conditions . The antibody shows human reactivity, making it suitable for studies utilizing human cells or tissues expressing ADGRG3/GPR97 .

How does ADGRG3/GPR97 function in inflammatory processes?

ADGRG3/GPR97 plays a significant role in inflammatory processes, particularly in neutrophils, through a complex mechanism involving multiple protein interactions. Research has revealed that GPR97 allosterically activates CD177-associated membrane proteinase 3 (mPR3) as part of a macromolecular complex that includes CD177, PAR2, and CD16b . This activation triggers inflammatory responses in neutrophils.

The mechanistic pathway involves:

• GPR97 binding to mPR3, albeit less efficiently than CD177

• GPR97 allosterically enhancing mPR3 enzymatic activity

• The activated mPR3 cleaving and activating PAR2 (a G protein-coupled receptor known for its function in inflammation)

• PAR2 activation leading to robust neutrophil activation and inflammatory responses

This cascade represents an adhesion GPCR-GPCR activation mechanism that contributes to neutrophil-mediated inflammatory responses. The GPR97-ECR (extracellular region) contains specific mPR3-binding domains that facilitate this interaction . This understanding provides important insights for researchers studying inflammatory and autoimmune diseases where neutrophil dysfunction may play a role.

What methodological considerations are important when using HRP-conjugated ADGRG3 antibodies in ELISA?

When employing HRP-conjugated ADGRG3 antibodies in ELISA, several methodological considerations are essential for obtaining reliable and reproducible results:

Antibody specificity verification:

• Confirm specificity using positive and negative controls expressing or lacking ADGRG3/GPR97

• Consider the epitope location (AA 98-244 for some commercially available antibodies) when designing experiments

• Be aware that the antibody targets recombinant Human Adhesion G protein-coupled receptor G3 protein at a specific amino acid range

Optimization protocols:

• Determine optimal antibody dilution through titration experiments (manufacturers note that "optimal working dilution should be determined by the investigator")

• Consider blocking conditions carefully to minimize background signal

• Assess potential cross-reactivity with other adhesion GPCRs due to structural similarities

• Validate detection limits and linear range specifically for your experimental system

Technical execution:

• Standardize sample preparation methods for consistency

• Include appropriate reference standards for quantitative analyses

• Consider the impact of protein-protein interactions on epitope accessibility, especially when studying GPR97 within its macromolecular complex

• Implement rigorous washing protocols to minimize background signal

Implementation of these methodological considerations will help ensure that experimental data generated using HRP-conjugated ADGRG3 antibodies in ELISA applications provides reliable insights into ADGRG3/GPR97 biology.

How can ADGRG3/GPR97 signaling be effectively studied using reporter assays?

Studying ADGRG3/GPR97 signaling through reporter assays represents an advanced approach to understanding this receptor's downstream signaling pathways. Recent research has developed a fusion protein platform that provides robust analysis of aGPCR signaling, including ADGRG3/GPR97 . This platform involves fusion of maltose-binding protein (MBP) with a linker containing a TEV cleavage site to the tethered agonist (TA) sequence at the amino-terminal end of the aGPCR signaling subunit.

Recommended reporter assay methodology:

• Utilize the MBP-CTF (C-terminal fragment) fusion platform for robust expression and signaling activity

• Implement multiple reporter systems, particularly:

  • Serum response element (SRE) reporter, which ADGRG3 has been confirmed to stimulate

  • cAMP response element (CRE) reporter, which represents a novel signal for ADGRG3 identified through this platform

• Conduct assays in engineered cell lines (such as HEK293T) for determining G protein subtype specificity

• Compare autonomous activity to peptide-induced activation to distinguish signaling mechanisms

Research has revealed that ADGRG3/GPR97 can induce both SRE and CRE reporters, indicating coupling to multiple G protein pathways . These findings expand our understanding of ADGRG3/GPR97 signaling beyond previous reports.

For comprehensive characterization, complementary approaches can include:

• $³⁵S$GTPγS binding assays

• Proximity-based recruitment assays

• Structure-function relationship studies through targeted mutations

This methodological framework allows for in-depth investigation of ADGRG3/GPR97 signaling mechanisms, G protein coupling preferences, and structure-function relationships.

What strategies can resolve contradictory data regarding ADGRG3/GPR97 G protein coupling specificity?

Resolving contradictory data regarding ADGRG3/GPR97 G protein coupling specificity requires systematic experimental approaches and careful data interpretation. Research on related adhesion GPCRs, such as CD97, has demonstrated selective coupling to G12/13 heterotrimers , while newer studies have uncovered additional signaling pathways for ADGRG3/GPR97.

Recommended resolution strategies:

Experimental approach diversification:

• Employ engineered cell lines lacking specific G protein subunits (such as HEKΔ6 cells lacking six Gα subunits) to precisely define coupling profiles

• Utilize multiple, complementary reporter systems that reflect different G protein signaling pathways

• Implement the MBP-CTF fusion platform, which has identified novel signals for ADGRG3, including both SRE and CRE reporter activation

• Combine cellular assays with direct biochemical measurements such as $³⁵S$GTPγS binding

Contextual factors evaluation:

• Assess the impact of expression system (cell type) on coupling preferences

• Consider the influence of the receptor's activation state (basal vs. stimulated)

• Evaluate the effect of the receptor's molecular environment, including potential interaction partners

• Account for methodology-dependent biases in detecting different G protein couplings

Data integration framework:

• Compare data across multiple experimental systems while accounting for technical limitations

• Consider signaling profiles in physiologically relevant contexts (e.g., neutrophils for inflammatory pathways)

• Differentiate between primary and secondary coupling events in signaling cascades

• Develop comprehensive models that integrate direct and indirect signaling effects

By implementing these strategies, researchers can address contradictory findings regarding ADGRG3/GPR97 G protein coupling specificity and develop a more nuanced understanding of this receptor's signaling capabilities across different cellular contexts.

How should experiments be designed to study interactions between ADGRG3/GPR97 and partner proteins?

Designing robust experiments to study ADGRG3/GPR97 interactions with partner proteins requires multiple complementary approaches. Based on research identifying GPR97's involvement in a macromolecular complex with CD177, mPR3, PAR2, and CD16b in neutrophils , the following experimental design strategies are recommended:

Protein-protein interaction verification methods:

• FACS-based ligand-binding assays in expression systems such as HEK-293T cells co-expressing ADGRG3/GPR97 and potential partners

• ELISA-like protein-protein binding assays using purified components

• Co-immunoprecipitation followed by immunoblotting to detect native protein complexes

• Proximity ligation assays for detecting interactions in intact cells or tissues

Binding domain mapping approaches:

• Generation of domain-swapped fusion proteins (as demonstrated with GPR97 and GPR56 GAIN domains)

• Development of deletion constructs to identify critical binding regions

• Site-directed mutagenesis of specific residues within candidate binding domains

• Crystallographic analysis of interaction interfaces when feasible

Functional consequence evaluation:

• Enzymatic activity assays (particularly relevant for studying GPR97's allosteric activation of mPR3)

• Downstream signaling readouts including calcium flux, ERK phosphorylation, or reporter gene activation

• Cellular functional assays relevant to neutrophil biology (e.g., migration, degranulation, NETs formation)

• Ex vivo analysis using primary cells expressing endogenous levels of interaction partners

By implementing this multi-faceted experimental approach, researchers can comprehensively characterize the interactions between ADGRG3/GPR97 and its partner proteins, mapping both structural requirements and functional consequences of these interactions.

What are the optimal methodologies for comparing ADGRG3/GPR97 expression across different cell types?

Comparing ADGRG3/GPR97 expression across different cell types requires careful selection of complementary methodologies to ensure accurate and comprehensive characterization. Based on the available research, the following methodological approaches are recommended:

mRNA expression analysis:

• qRT-PCR with carefully validated primer sets specific to ADGRG3/GPR97

• RNA sequencing for comprehensive transcriptomic profiling

• Single-cell RNA sequencing to identify cell-specific expression patterns within heterogeneous populations

• Temporal analysis during cellular processes (e.g., differentiation stages as demonstrated in adipogenesis studies)

Protein expression evaluation:

• Western blotting using specific antibodies (including HRP-conjugated ADGRG3 antibodies for direct detection)

• Flow cytometry for cell surface expression quantification

• Immunofluorescence or immunohistochemistry for localization studies

• ELISA for quantitative comparison of expression levels

Experimental design considerations:

• Include appropriate positive and negative control cell types

• Standardize cell culture conditions to minimize variability

• Account for potential differences in receptor trafficking or post-translational modifications

• Consider examining expression under different stimulation conditions relevant to ADGRG3/GPR97 function

Data normalization and comparison framework:

• Normalize protein expression to appropriate housekeeping proteins

• Implement statistical methods suitable for multiple group comparisons

• Correlate mRNA and protein expression data to identify potential post-transcriptional regulation

• Present data in standardized formats (e.g., fold-change relative to a reference cell type)

This comprehensive methodological approach enables reliable comparison of ADGRG3/GPR97 expression across diverse cell types, providing insights into tissue-specific functions and potential roles in physiological or pathological processes.

How can functional studies of ADGRG3/GPR97 be designed to identify potential therapeutic targets?

Designing functional studies of ADGRG3/GPR97 to identify potential therapeutic targets requires strategic experimental approaches spanning from molecular mechanisms to disease relevance. Given GPR97's role in inflammatory processes in neutrophils and its involvement in signaling pathways , the following experimental design framework is recommended:

Mechanistic characterization approaches:

• Structure-function studies using the MBP-CTF fusion platform to identify critical domains and residues

• G protein coupling profiling in engineered cell lines to delineate downstream signaling pathways

• Allosteric modulator screening to identify compounds that can selectively modify receptor activity

• Molecular dynamics simulations to predict druggable binding pockets

Cellular function evaluation:

• Neutrophil activation assays measuring endpoints such as reactive oxygen species production, degranulation, and NETosis

• Migration and adhesion assays to assess GPR97's role in neutrophil recruitment and tissue infiltration

• Co-culture systems to investigate neutrophil-endothelial interactions, particularly in inflammatory contexts

• Ex vivo studies using neutrophils from patients with inflammatory or autoimmune conditions

Validation in disease models:

• Genetic approaches using CRISPR/Cas9 to generate cellular or animal models with modified ADGRG3/GPR97

• Pharmacological interventions using identified modulators in disease-relevant models

• Patient-derived samples to correlate ADGRG3/GPR97 function with disease severity or treatment response

• Tissue-specific conditional knockout models to delineate context-dependent functions

Therapeutic potential assessment:

• High-throughput screening for compounds that modulate the GPR97-mPR3-PAR2 activation pathway

• Development of blocking antibodies targeting specific functional domains of ADGRG3/GPR97

• Design of decoy peptides that can interfere with the GPR97-containing macromolecular complex

• Biomarker identification studies correlating ADGRG3/GPR97 activity with disease progression or therapeutic response

This comprehensive experimental approach can identify potential therapeutic targets within the ADGRG3/GPR97 signaling axis, particularly for inflammatory and autoimmune conditions where neutrophil dysfunction plays a pathogenic role.

How should researchers interpret contradictory results in ADGRG3/GPR97 binding studies?

Interpreting contradictory results in ADGRG3/GPR97 binding studies requires systematic analysis of methodological differences and biological context. Research has demonstrated that GPR97 binds to PR3 specifically but with lower efficiency compared to CD177 . When encountering contradictory binding data, researchers should consider the following interpretative framework:

Methodological factors assessment:

• Compare binding assay formats (e.g., FACS-based vs. ELISA-like protein-protein binding assays)

• Evaluate protein expression systems and their impact on post-translational modifications

• Analyze protein purification methods and their effect on native conformation

• Consider detection method sensitivity and potential epitope masking effects

Binding parameter comparison:

• Distinguish between binding affinity (Kd) and binding capacity (Bmax) measurements

• Compare association and dissociation kinetics when available

• Assess the impact of concentration ranges used in different studies

• Evaluate temperature, pH, and buffer composition differences between experimental setups

Biological context consideration:

• Analyze differences between recombinant systems and endogenous expression contexts

• Consider the impact of macromolecular complex formation on binding properties

• Evaluate the role of allosteric modulators present in different experimental systems

• Account for cell type-specific factors that might influence receptor conformation or accessibility

Resolution strategies:

• Implement multiple, complementary binding assay formats within the same study

• Characterize binding under standardized conditions with systematic parameter variation

• Use competition assays to determine binding site overlap or distinctness

• Perform structure-function studies to map binding interfaces and reconcile apparent contradictions

By applying this interpretative framework, researchers can systematically address contradictory results in ADGRG3/GPR97 binding studies, potentially revealing nuanced aspects of receptor-ligand interactions that depend on specific methodological or biological contexts.

What are common technical challenges in ADGRG3 antibody experiments and how can they be overcome?

Working with ADGRG3/GPR97 antibodies presents several technical challenges that can impact experimental outcomes. Based on available research and general antibody methodology principles, the following challenges and solutions are most relevant:

Specificity verification challenges:

• Cross-reactivity with related adhesion GPCRs due to sequence homology

• Non-specific binding in complex samples or certain cell types

• Epitope masking in native complexes involving ADGRG3/GPR97

Solutions:

• Validate antibody specificity using knockout/knockdown controls

• Perform pre-absorption controls with immunizing peptide

• Test multiple antibodies targeting different epitopes

• Compare reactivity patterns across tissues known to express or lack ADGRG3/GPR97

Signal detection optimization:

• Insufficient signal strength in samples with low expression levels

• High background interfering with specific signal detection

• Variable HRP activity affecting signal consistency

Solutions:

• Optimize antibody concentration through careful titration experiments

• Implement enhanced blocking protocols using carrier proteins

• Consider signal amplification systems for low-abundance targets

• Standardize incubation times and temperatures for enzymatic reactions

Sample preparation considerations:

• Protein denaturation affecting conformational epitopes

• Inefficient cell lysis leading to incomplete protein extraction

• Degradation during sample processing

Solutions:

• Use mild lysis conditions for membrane proteins like ADGRG3/GPR97

• Include appropriate protease inhibitors in all buffers

• Minimize freeze-thaw cycles of samples and antibody reagents

• Consider native condition preservation for conformational epitopes

Experimental design improvements:

• Include comprehensive controls (positive, negative, isotype)

• Standardize sample handling procedures

• Validate results with complementary detection methods

• Consider the impact of experimental conditions on ADGRG3/GPR97 expression levels or conformational states

By addressing these common technical challenges with the proposed solutions, researchers can enhance the reliability and reproducibility of experiments using ADGRG3/GPR97 antibodies, particularly those conjugated to HRP for direct detection applications.

How can researchers distinguish between direct and indirect effects in ADGRG3/GPR97 signaling studies?

Distinguishing between direct and indirect effects in ADGRG3/GPR97 signaling studies represents a critical challenge, especially given its involvement in complex signaling networks and macromolecular complexes . The following methodological framework provides strategies to address this challenge:

Temporal resolution approaches:

• Implement high-resolution time-course experiments to identify sequential signaling events

• Utilize rapid kinetic measurements to capture immediate receptor activation responses

• Compare activation kinetics across different downstream pathways to identify primary vs. secondary events

• Apply mathematical modeling to deconvolute complex signaling dynamics

Pharmacological intervention strategies:

• Use selective inhibitors of specific downstream effectors to block secondary signaling events

• Implement dose-response studies with selective agonists or antagonists

• Apply pathway-specific inhibitors to determine signaling route dependencies

• Develop and utilize biased ligands that preferentially activate specific signaling pathways

Genetic manipulation approaches:

• Generate cell lines with CRISPR/Cas9-mediated knockout of potential signaling intermediates

• Develop receptor mutants that selectively couple to specific G protein subtypes

• Use dominant-negative constructs to inhibit specific signaling branches

• Implement inducible expression systems to control signaling component availability

Biochemical validation methods:

• Perform direct G protein activation assays such as $³⁵S$GTPγS binding

• Implement proximity-based assays (BRET, FRET) to detect immediate protein-protein interactions

• Use cell-free reconstitution systems with purified components to verify direct interactions

• Apply label-transfer approaches to capture transient signaling complexes

Integrated analytical framework:

• Combine multiple methodological approaches within the same study

• Compare signaling profiles across different cell types with varying expression of potential intermediates

• Develop network models that predict direct vs. indirect signaling relationships

• Validate key findings in physiologically relevant systems such as primary neutrophils

This comprehensive methodological framework enables researchers to systematically distinguish between direct and indirect effects in ADGRG3/GPR97 signaling studies, providing clearer insights into the receptor's primary signaling mechanisms and their downstream consequences.

What emerging technologies show promise for advancing ADGRG3/GPR97 research?

Several emerging technologies show significant promise for advancing ADGRG3/GPR97 research, potentially overcoming current limitations and enabling new insights into this receptor's biology. Based on recent developments in GPCR research and specific findings related to adhesion GPCRs, the following technologies are particularly promising:

Advanced structural biology approaches:

• Cryo-electron microscopy (cryo-EM) for determining full-length ADGRG3/GPR97 structures in different activation states

• Single-particle analysis of the macromolecular complex formed with CD177, mPR3, PAR2, and CD16b

• Hydrogen-deuterium exchange mass spectrometry (HDX-MS) to map dynamic conformational changes during activation

• Advanced computational modeling integrating experimental structural constraints

Innovative signaling detection methods:

• CRISPR-based transcriptional reporters for endogenous pathway activation monitoring

• Genetically-encoded biosensors for real-time, spatially-resolved signaling visualization

• Single-molecule imaging of receptor dynamics and interactions in live cells

• Nanobody-based probes for conformation-specific receptor detection

Genetic engineering advancements:

• Base editing and prime editing for precise introduction of specific mutations

• CRISPR activation/inhibition systems for endogenous expression modulation

• Orthogonal receptor-effector pairs for pathway-specific signaling analysis

• Tissue-specific and inducible manipulation in complex model systems

System-level analysis technologies:

• Spatial transcriptomics and proteomics to map ADGRG3/GPR97 expression and signaling in tissue contexts

• Multi-omics integration approaches to correlate receptor activity with global cellular changes

• Advanced bioinformatics methods for predicting receptor-ligand interactions and signaling networks

• Organ-on-chip technologies for modeling GPR97 function in inflammatory microenvironments

Therapeutic development platforms:

• DNA-encoded chemical libraries for high-throughput screening of modulators

• Fragment-based drug discovery optimized for adhesion GPCR targets

• Structure-based virtual screening utilizing emerging structural data

• Antibody engineering platforms for developing function-modifying antibodies

These emerging technologies have the potential to significantly advance ADGRG3/GPR97 research by providing unprecedented insights into receptor structure, dynamics, signaling mechanisms, physiological functions, and therapeutic targeting opportunities.