ADGRG1 Antibody

What is ADGRG1 and why is it significant in neurological research?

ADGRG1 (adhesion G protein-coupled receptor G1), previously known as GPR56, is a 693-amino acid protein belonging to the G-protein coupled receptor 2 family, LN-TM7 subfamily . It functions as a membrane-associated and secreted protein with documented glycosylation sites . ADGRG1 has gained significant research attention due to its specific expression in yolk-sac-derived microglia and its critical role in modulating protective microglial responses to amyloid deposition in Alzheimer's disease (AD) .

Recent transcriptomic studies have revealed that ADGRG1 is one of the top 300 significantly altered genes in AD according to GWAS data, suggesting its potential as a therapeutic target . Functionally, this receptor is essential for efficient Aβ phagocytosis, a crucial process for limiting amyloid plaque burden . Research has demonstrated that ADGRG1 deficiency compromises microglial association with amyloid plaques and their phagocytic capacity, leading to increased amyloid deposition, exacerbated neuropathology, and accelerated cognitive decline in mouse models .

What applications are ADGRG1 antibodies most commonly used for in research?

ADGRG1 antibodies are utilized across multiple research applications, with particular emphasis on techniques that investigate protein expression, localization, and function in neurological contexts. Common applications include:

• Western Blotting (WB): For detecting and quantifying ADGRG1 protein expression levels in tissue or cell lysates .

• Immunohistochemistry (IHC): Particularly on paraffin-embedded sections (IHC-p) to visualize ADGRG1 distribution in brain tissues and study its localization in specific cell types .

• Immunocytochemistry (ICC) and Immunofluorescence (IF): To examine subcellular localization of ADGRG1 in cultured cells, including primary microglia and cell lines .

• Flow Cytometry (FCM): For quantifying ADGRG1 expression in individual cells and isolating ADGRG1-positive cell populations .

• ELISA: For quantitative measurement of ADGRG1 in biological samples .

Studies investigating microglial functions in neurodegenerative diseases frequently employ these techniques to correlate ADGRG1 expression with phagocytic capacity, particularly in the context of Alzheimer's disease research .

How can I validate the specificity of an ADGRG1 antibody for my research?

Validating antibody specificity is crucial for ensuring reliable research outcomes. For ADGRG1 antibodies, consider implementing these validation approaches:

• Positive and Negative Controls:

  • Positive: Use tissues or cells known to express ADGRG1 (e.g., microglia in brain tissue)

  • Negative: Include ADGRG1 knockout samples as demonstrated in the literature using CRISPR/Cas9-based techniques

• Blocking Peptide Analysis: Pre-incubate the antibody with a synthetic peptide corresponding to the immunogen to confirm specific binding

• Multiple Antibody Validation: Compare staining patterns using antibodies targeting different epitopes of ADGRG1

• Molecular Weight Verification: In Western blots, confirm the detected band corresponds to the expected 693-amino acid protein size, accounting for potential post-translational modifications like glycosylation

• Gene Silencing Validation: Compare antibody staining in cells with and without ADGRG1 knockdown (siRNA or CRISPR)

The literature describes CRISPR/Cas9-based knockout validation approaches for ADGRG1, using gRNA sequences such as 5'-ACACTCTTCCAGAGGACGAA-3' targeting exon 7 of the ADGRG1 gene locus . This technique can be adapted to validate antibody specificity by confirming absence of signal in knockout cells.

How should I design experiments to investigate ADGRG1's role in Aβ phagocytosis?

Designing experiments to investigate ADGRG1's role in Aβ phagocytosis requires a multi-faceted approach that combines in vitro and in vivo methodologies:

• In Vitro Phagocytosis Assays:

  • Primary microglia or human embryonic stem cell (hESC)-derived microglia cultures with ADGRG1 knockout or knockdown

  • Fluorescently labeled Aβ to track internalization

  • Live-cell imaging to monitor phagocytosis dynamics

  • Quantification of internalized Aβ using flow cytometry or confocal microscopy

• In Vivo Models:

  • Generate microglial-specific ADGRG1 knockout models using Cre-lox systems (e.g., Adgrg1 fl/fl;Cx3cr1Cre/+ or inducible systems like Adgrg1 fl/fl;P2ry12CreER/+)

  • Cross these models with Alzheimer's disease mouse models (e.g., 5xFAD)

  • Assess amyloid plaque burden through immunostaining with antibodies against Aβ (e.g., MOAB2 or H31L21)

  • Quantify microglial-plaque interactions by measuring microglial density within a 30-μm radius of plaques

• Mechanistic Studies:

  • Transcriptomic analysis to identify differentially expressed genes related to phagocytosis

  • Examine lysosomal function using markers like LAMP1

  • Assess changes in expression of phagocytosis-related genes (e.g., CD68, CTSD, CTSB)

Research has shown that ADGRG1-deficient microglia exhibit reduced association with amyloid plaques and compromised phagocytic capacity, resulting in increased amyloid deposition . This suggests experimental readouts should include both direct measures of phagocytosis and secondary effects on plaque burden.

What are the best approaches for analyzing ADGRG1 expression in different microglial activation states?

Analyzing ADGRG1 expression across different microglial activation states requires an integrated approach combining transcriptomic, protein-level, and functional analyses:

• Single-cell/Single-nucleus RNA Sequencing:

  • Enables identification of distinct microglial subclusters based on transcriptomic profiles

  • Can reveal how ADGRG1 expression correlates with homeostatic markers (e.g., Tmem119, P2ry12) or disease-associated microglia (DAM) markers

  • Allows for unbiased identification of co-regulated gene networks

• Protein-level Analysis:

  • Immunofluorescence co-labeling of ADGRG1 with markers of different microglial states:

    • Homeostatic: P2ry12, Cx3cr1, Tmem119

    • DAM: Tyrobp, Apoe, Trem2

    • Phagocytic: CD68, C1qa/b/c, Cd81

  • Flow cytometry for quantitative assessment of ADGRG1 levels in different microglial populations

• Spatial Context Assessment:

  • Analyze ADGRG1 expression in microglia proximal to vs. distal from amyloid plaques

  • 3D reconstruction of immunostained tissues to evaluate spatial relationships

Research has demonstrated that microglial ADGRG1 deletion in 5xFAD mice leads to a transcriptomic profile characterized by downregulation of both homeostatic and phagocytic genes . Unlike Trem2 deletions, which prevent microglia from transitioning to DAM states, ADGRG1 deletion shifts microglial function while still allowing DAM progression but alters subpopulations within this state . This suggests that ADGRG1 plays a unique role in regulating microglial functional states beyond simple activation markers.

How can I correlate ADGRG1 levels with Alzheimer's disease progression in human samples?

Correlating ADGRG1 levels with Alzheimer's disease progression in human samples requires careful consideration of tissue sources, disease staging, and analytical approaches:

• Patient Cohort Selection:

  • Include individuals across the disease spectrum: cognitively normal, mild cognitive impairment (MCI), and established AD

  • Stratify samples according to established pathological staging (e.g., Thal phase, Braak stage)

  • Consider genetic factors like APOE genotype that might influence ADGRG1 function

• Tissue Analysis Approaches:

  • Immunohistochemistry: Use validated antibodies like CG4 (a monoclonal antibody against human ADGRG1) with microglia markers (e.g., Iba1)

  • Focus on brain regions vulnerable to AD pathology, such as the middle temporal gyrus

  • Quantify ADGRG1 signal intensity in microglia proximal to Aβ deposits

• Transcriptomic Integration:

  • Analyze existing single-nucleus RNA-seq datasets from AD brain tissue

  • Perform correlation analyses between ADGRG1 and phagocytosis-related genes

  • Investigate relationships with known AD risk genes

Research has shown that microglia from individuals with mild cognitive impairment due to AD (MCI) exhibit more prominent ADGRG1 (CG4-positive) signals compared to those from AD patients, suggesting a potential resilient function of microglial ADGRG1 in early disease stages . Additionally, transcriptomic meta-analysis across 590 human brain tissues revealed that microglial ADGRG1 expression positively correlates with phagocytosis-related genes (CD68, CTSD, CTSB) and negatively correlates with SSH2 and INSR . These findings suggest that temporal dynamics of ADGRG1 expression may be important in disease progression.

What are the optimal fixation and permeabilization protocols for ADGRG1 immunostaining in brain tissue?

Optimizing fixation and permeabilization for ADGRG1 immunostaining requires balancing epitope preservation with tissue penetration:

• Tissue Preparation:

  • For formalin-fixed paraffin-embedded (FFPE) sections (14-μm thickness):

    • Perform antigen retrieval for 5 minutes at 95°C using an antigen retrieval buffer (e.g., BD Pharmingen, 550524)

    • For enhanced permeability with anti-ADGRG1 (CG4) antibody, treat sections with Protease III (ACD) at 40°C for 15 minutes before blocking

• Blocking Conditions:

  • Block in buffer containing 5% goat serum, 1% bovine serum albumin (BSA), and 0.03% Triton X-100 in PBS at room temperature for 1 hour

• Antibody Incubation:

  • Primary antibody: Incubate overnight at 4°C with appropriate dilution (e.g., anti-ADGRG1/CG4 at 1:200)

  • Secondary antibody: Apply at 1:500 dilution at room temperature for 2 hours

• Multi-labeling Considerations:

  • For co-staining with microglial markers, use anti-Iba1 (1:500)

  • For amyloid plaque visualization, use anti-Aβ antibodies (MOAB2 or H31L21, 1:1000)

This protocol has been successfully used to visualize ADGRG1 in human brain tissue, particularly for studying its expression in microglia in the context of Alzheimer's disease . The specific antigen retrieval and proteolytic treatment steps are crucial for accessing the ADGRG1 epitope while maintaining tissue integrity.

What controls should I include when performing Western blots with ADGRG1 antibodies?

Western blot analysis of ADGRG1 requires careful consideration of controls to ensure specificity and reliability:

• Essential Controls:

  • Positive Control: Lysate from tissues or cells known to express ADGRG1 (e.g., microglial cell lines, brain tissue)

  • Negative Control: Lysate from ADGRG1 knockout models or cells with CRISPR/Cas9-mediated ADGRG1 deletion

  • Loading Control: Antibodies against housekeeping proteins (e.g., GAPDH, β-actin) to normalize expression levels

  • Molecular Weight Marker: To confirm the detected band corresponds to the expected size of ADGRG1

• Specificity Controls:

  • Peptide Competition: Pre-incubation of antibody with immunogen peptide should abolish specific ADGRG1 band

  • Secondary Antibody Only: Omitting primary antibody to assess non-specific binding of secondary antibody

• Technical Considerations:

  • Account for post-translational modifications that might affect molecular weight (e.g., glycosylation sites reported for ADGRG1)

  • Consider detergent selection for membrane protein extraction (ADGRG1 is a membrane-associated protein)

  • For quantitative analysis, use gradient gels (4-12%) to improve resolution

• Experimental Controls:

  • Include samples with experimentally modulated ADGRG1 levels (e.g., overexpression, knockdown)

  • When studying disease models, include both affected and unaffected tissue samples

For ADGRG1 knockout models, CRISPR/Cas9-based non-homology end joining with guide RNA sequence (5'-ACACTCTTCCAGAGGACGAA-3') targeting exon 7 of the ADGRG1 gene locus has been successfully used to generate negative controls . When validating knockouts, confirm by Sanger sequencing and check for potential off-target effects.

How can I quantify changes in ADGRG1 expression in microglia surrounding amyloid plaques?

Quantifying ADGRG1 expression in microglia around amyloid plaques requires sophisticated imaging and analysis approaches:

• Tissue Preparation and Staining:

  • Triple immunofluorescence staining for:

    • ADGRG1 (e.g., CG4 antibody, 1:200)

    • Microglial marker (e.g., Iba1, 1:500)

    • Amyloid plaques (e.g., anti-Aβ MOAB2 or H31L21, 1:1000)

• Imaging Approaches:

  • Confocal microscopy with z-stack acquisition to capture the three-dimensional relationship between microglia and plaques

  • High-resolution imaging (at least 63× objective) for detailed subcellular localization

  • Consider super-resolution techniques for finer detail of receptor distribution

• Quantification Methods:

  • Spatial Analysis:

    • Define concentric regions of interest (ROIs) at different distances from plaque borders (e.g., 0-10 μm, 10-20 μm, 20-30 μm)

    • Count Iba1+ cells within each ROI

    • Measure ADGRG1 fluorescence intensity in Iba1+ cells in each ROI

  • Morphological Analysis:

    • Perform 3D reconstruction of microglial cells using appropriate software

    • Analyze morphological parameters (process length, branching, cell body size)

    • Correlate morphology with ADGRG1 expression levels

• Statistical Approach:

  • Compare ADGRG1 expression in microglia at different distances from plaques

  • Analyze differences between experimental groups (e.g., control vs. ADGRG1-deficient models)

  • Account for plaque size and density in the analysis

Research has shown that the number of microglia within a 30-μm radius from plaques was significantly reduced in both constitutive and inducible ADGRG1 knockout models compared to controls . This methodology can be adapted to quantify not only microglial numbers but also ADGRG1 expression levels in the cells present, providing insight into the relationship between receptor expression and microglial plaque association.

How does ADGRG1 expression in human microglia compare between Alzheimer's disease patients and controls?

ADGRG1 expression in human microglia shows distinct patterns across the Alzheimer's disease continuum, with important implications for understanding disease pathophysiology:

• Expression Patterns Across Disease Stages:

  • Studies examining human middle temporal gyrus tissue (a region vulnerable to AD pathological changes) revealed that microglia from individuals with mild cognitive impairment (MCI) due to AD exhibit more prominent CG4-positive (ADGRG1) signals compared to those from established AD patients

  • This suggests that microglial ADGRG1 may serve a protective function in early disease stages, potentially contributing to disease resilience

  • ADGRG1 expression appears to decline as disease progresses from MCI to established AD

• Correlation with Functional Markers:

  • Transcriptomic meta-analysis across 590 human brain tissues demonstrated that microglial ADGRG1 expression positively correlates with phagocytosis-related genes:

    • CD68 (lysosomal marker associated with phagocytosis)

    • CTSD and CTSB (lysosomal proteases)

  • ADGRG1 negatively correlates with SSH2 and INSR

  • These correlations align with findings from mouse models, suggesting conserved functional relationships

• Genetic Associations:

  • GWAS data analysis identified ADGRG1 as one of the top 300 significantly altered genes in AD

  • Comparison of microglial differentially expressed genes from single-nucleus RNA-seq data with known human AD risk genes revealed:

    • 24 AD risk genes overlapped with genes upregulated in control microglia

    • 97 AD risk genes overlapped with genes upregulated in ADGRG1-deficient microglia

  • This substantial increase suggests that ADGRG1 deficiency shifts microglial gene expression toward a profile associated with increased AD risk

These findings highlight the potential role of ADGRG1 in modulating disease resilience through microglial functional properties, particularly in early disease stages. The temporal dynamics of ADGRG1 expression may be critical for understanding the transition from protective to dysfunctional microglial responses in AD progression.

What techniques are most effective for studying ADGRG1-mediated signaling pathways in microglia?

Investigating ADGRG1-mediated signaling pathways in microglia requires a combination of molecular, cellular, and functional approaches:

• Receptor Activation Studies:

  • Use of natural ligands or antibodies that can activate ADGRG1

  • Development of small molecule activators or inhibitors

  • CRISPR-based mutagenesis of specific domains to study structure-function relationships

• Downstream Signaling Analysis:

  • Phosphoproteomic Analysis:

    • Identify phosphorylation events triggered by ADGRG1 activation

    • Use phospho-specific antibodies to monitor activation of suspected downstream effectors

  • G-protein Coupling Studies:

    • BRET/FRET assays to monitor G-protein coupling

    • Selective G-protein inhibitors to dissect pathway contributions

  • Secondary Messenger Assays:

    • cAMP, calcium imaging, or IP3 measurement following receptor activation

    • Real-time monitoring using fluorescent biosensors

• Functional Pathway Validation:

  • Pharmacological Approach:

    • Pathway-specific inhibitors to block suspected signaling components

    • Measure effects on phagocytosis, migration, or inflammatory responses

  • Genetic Approach:

    • siRNA or CRISPR/Cas9 to knock down components of putative signaling pathways

    • Rescue experiments in ADGRG1-deficient cells by expressing downstream effectors

• Transcriptomic Response:

  • RNA-seq after ADGRG1 activation to identify regulated gene networks

  • Time-course analysis to distinguish primary from secondary responses

  • Comparison with known microglial activation pathways (e.g., TREM2-SYK signaling)

Research has shown that unlike TREM2 deletions, where microglia fail to fully transition from homeostatic to disease-associated microglia (DAM) states, ADGRG1 deletion shifts microglial function without affecting DAM progression but alters subpopulations within this state . This suggests that ADGRG1 signaling may regulate specific functional aspects of microglial responses rather than broadly controlling activation state transitions.

How can I troubleshoot non-specific binding or high background when using ADGRG1 antibodies in immunohistochemistry?

Troubleshooting non-specific binding and high background issues with ADGRG1 antibodies requires a systematic approach to identify and address the source of the problem:

• Antibody-Related Issues:

  • Titration Optimization: Test a range of antibody dilutions (e.g., 1:100, 1:200, 1:500, 1:1000) to find the optimal signal-to-noise ratio

  • Incubation Conditions: Try shorter incubation times at room temperature instead of overnight at 4°C

  • Validate Specificity: Test the antibody on ADGRG1 knockout tissues as negative controls

  • Alternative Antibodies: Compare results using antibodies targeting different epitopes of ADGRG1

• Tissue Preparation Optimization:

  • Fixation Parameters: Overfixation can cause high background; adjust fixation times

  • Antigen Retrieval: For ADGRG1 staining, optimize antigen retrieval by testing:

    • Heat-induced epitope retrieval at 95°C for 5 minutes

    • Enzymatic retrieval using Protease III treatment at 40°C for 15 minutes

  • Blocking Improvements: Increase blocking reagent concentration (e.g., from 5% to 10% serum) or try alternative blockers like protein-free blockers

• Protocol Modifications:

  • Additional Blocking Steps:

    • Include avidin/biotin blocking if using biotinylated secondary antibodies

    • Add mouse IgG blocking for mouse tissues when using mouse primary antibodies

  • Washing Steps: Increase washing duration and frequency between steps

  • Detection System: Switch between direct and indirect detection methods

  • Endogenous Peroxidase Quenching: If using HRP-based detection, ensure complete quenching of endogenous peroxidase

• Advanced Techniques for Difficult Samples:

  • Tyramide Signal Amplification: For weak ADGRG1 signals while minimizing background

  • Fluorescence Background Reduction: Include an autofluorescence quenching step

  • Sequential Double Staining: Perform complete single stainings sequentially rather than coincubating primary antibodies

The search results indicate that researchers have successfully used specific protocols for ADGRG1 staining, including antigen retrieval for 5 minutes at 95°C, blocking with 5% goat serum and 1% BSA, and enhancing permeability with Protease III treatment before blocking specifically for the CG4 anti-ADGRG1 antibody . Adapting these validated approaches can help resolve background issues.

What are the key considerations when using ADGRG1 antibodies for co-localization studies with other microglial markers?

Successful co-localization studies with ADGRG1 and other microglial markers require careful attention to several technical aspects:

• Antibody Compatibility Planning:

  • Host Species Selection: Choose primary antibodies raised in different host species to avoid cross-reactivity

  • Isotype Consideration: If antibodies are from the same species, use different isotypes and isotype-specific secondary antibodies

  • Application Validation: Verify that each antibody performs well in multiplexed staining conditions

• Epitope Accessibility Optimization:

  • Sequential Antigen Retrieval: Different markers may require different retrieval methods

    • For ADGRG1 (CG4 antibody), a combination of heat-based antigen retrieval (95°C for 5 minutes) and Protease III treatment (40°C for 15 minutes) has been effective

  • Order of Antibody Application: Test sequential versus simultaneous application of primary antibodies

• Imaging and Detection Strategy:

  • Fluorophore Selection: Choose fluorophores with minimal spectral overlap

  • Controls for Bleed-through: Include single-stained samples to set imaging parameters

  • Sequential Scanning: For confocal microscopy, use sequential rather than simultaneous scanning

  • Objective Selection: Use high-NA objectives (1.3 or higher) for optimal resolution

• Validated Marker Combinations:

  • Microglial Markers Compatible with ADGRG1:

    • Iba1 (1:500) - general microglial marker

    • P2ry12, Tmem119 - homeostatic microglial markers

    • CD68, LAMP1 (1:200) - lysosomal/phagocytic markers

  • Plaque Co-localization: Anti-Aβ antibodies (MOAB2 or H31L21, 1:1000)

• Quantitative Analysis Approaches:

  • Manders' Overlap Coefficient: To quantify the degree of co-localization

  • Distance Analysis: Measure the proximity between ADGRG1 and other markers

  • 3D Reconstruction: For volumetric analysis of co-localization in tissue sections

Research has shown that microglia around amyloid plaques exhibit altered ADGRG1 expression, making co-localization studies particularly relevant for understanding the relationship between ADGRG1 and microglial function in AD . Studies have successfully used combinations of ADGRG1, Iba1, and amyloid markers to quantify microglial responses within a 30-μm radius from plaques , demonstrating the feasibility of these co-localization approaches.