ADGRA2 Antibody

What is ADGRA2 and why is it important in research?

ADGRA2, formerly known as GPR124 or TEM5, is an adhesion G protein-coupled receptor that functions as a key regulator of cerebrovascular development in vertebrates. It works together with the GPI-anchored glycoprotein Reck to stimulate Wnt7-dependent Wnt/β-catenin signaling, promoting brain vascular invasion in an endothelial cell-autonomous manner . The protein contains multiple protein-protein interaction domains, including leucine-rich repeats (LRRs) that are critical for proper trafficking to the plasma membrane . Understanding ADGRA2 function has significant implications for developmental biology and potentially for pathologies involving vascular development.

What species reactivity should I consider when selecting an ADGRA2 antibody?

Currently available commercial ADGRA2 antibodies primarily show reactivity against human and mouse ADGRA2 . When selecting an antibody, consider the evolutionary conservation of your target epitope and confirm the documented cross-reactivity for your species of interest. For zebrafish or other model organisms, additional validation may be necessary as standard commercial antibodies may not have been tested against these species .

What applications are ADGRA2 antibodies validated for?

Commercial ADGRA2 antibodies are typically validated for Western blot (WB), immunofluorescence (IF), immunocytochemistry (ICC), and enzyme-linked immunosorbent assay (ELISA) . The specific applications validated vary by supplier and antibody clone. For example, Boster Bio's A30802 antibody is guaranteed for ELISA, IF, ICC, and WB applications , while Atlas Antibodies validates their antibodies for immunohistochemistry (IHC), ICC-IF, and WB .

What controls should I include when using ADGRA2 antibodies?

At minimum, include a negative control (samples known to lack ADGRA2 expression) and a positive control (samples with confirmed ADGRA2 expression). For advanced experimental designs, consider including CRISPR/Cas9-engineered ADGRA2 knockout cells as a gold-standard negative control . Pre-adsorption of the antibody with the immunizing peptide (if available) can serve as a specificity control. For experiments involving Reck-ADGRA2 interactions, isolated expression of each protein may serve as important comparative controls, as the proteins have been shown to traffic independently to the plasma membrane .

What is the expected molecular weight for ADGRA2 in Western blot analyses?

There is a notable discrepancy between the calculated and observed molecular weights of ADGRA2. The calculated molecular weight is approximately 142.647 kDa , while the observed molecular weight in Western blot is typically around 72 kDa . This discrepancy could be due to post-translational modifications, protein processing (including autoproteolysis at the GPCR proteolytic site), or other factors affecting electrophoretic mobility. When performing Western blot analysis, be prepared to observe bands at both molecular weights, depending on the epitope recognized by your antibody and the sample preparation conditions.

How can I mitigate non-specific binding when using ADGRA2 antibodies?

Non-specific binding can be reduced through several approaches. First, optimize blocking conditions using 3-5% BSA or milk in TBS-T, recognizing that the optimal blocking agent may vary by application and tissue type. Second, ensure adequate washing steps (typically 3-5 washes of 5-10 minutes each) with appropriate buffers. Third, consider pre-adsorbing the antibody with tissues or cells lacking ADGRA2 expression. Finally, for particularly challenging samples, implement antigen retrieval methods for IHC/IF or adjust lysate preparation protocols for WB to ensure optimal epitope accessibility.

What sample preparation techniques are critical for detecting ADGRA2?

Since ADGRA2 is a transmembrane protein enriched at lateral cell borders and sites of cell-ECM contact , sample preparation is critical. For cell lysis prior to Western blot, use detergent-based buffers (such as RIPA or NP-40) supplemented with protease inhibitors. Avoid freeze-thaw cycles that might degrade the protein. For immunofluorescence applications, gentle fixation methods (such as 4% PFA for 10-15 minutes) followed by careful permeabilization (0.1-0.2% Triton X-100 for 5-10 minutes) generally yield better results than harsh fixation procedures.

How should I address epitope masking issues when detecting ADGRA2?

ADGRA2 functions within a complex with Reck and interacts with various proteins including Frizzled, LRP5, and LRP6 . These interactions may mask epitopes recognized by certain antibodies. To address this, consider using multiple antibodies targeting different regions of ADGRA2. Denaturing conditions in Western blot can expose hidden epitopes, while native conditions may be necessary for detecting specific conformational states. For tissues, optimized antigen retrieval methods (heat-induced or enzymatic) may expose masked epitopes in fixed specimens.

How can I investigate ADGRA2 trafficking using immunofluorescence techniques?

Research has shown that the leucine-rich repeat (LRR) domain determines proper ADGRA2 trafficking to the plasma membrane . To investigate trafficking, combine ADGRA2 antibody staining with markers for various cellular compartments (ER, Golgi, endosomes). Live-cell imaging with tagged constructs can complement fixed-cell immunofluorescence. For advanced studies, use temperature-controlled trafficking assays (e.g., 20°C block for Golgi retention) followed by release and antibody staining at different time points to track the protein's movement through the secretory pathway.

How can I detect the ADGRA2-Reck interaction in experimental systems?

The ADGRA2-Reck interaction occurs at the plasma membrane after independent trafficking of both proteins . To detect this interaction, consider co-immunoprecipitation using antibodies against either protein, followed by Western blot detection of the binding partner. Proximity ligation assays (PLA) offer in situ detection of protein interactions with spatial resolution. For live-cell studies, bimolecular fluorescence complementation (BiFC) or FRET-based approaches can be employed to visualize the dynamics of these interactions in real-time.

What approaches can be used to study the role of ADGRA2 in Wnt signaling?

ADGRA2 functions as a Wnt7-specific co-activator of canonical Wnt signaling . To study this role, TOPFlash reporter assays can measure β-catenin-dependent transcriptional activity in cells with manipulated ADGRA2 expression. Co-immunoprecipitation experiments can detect physical interactions between ADGRA2 and Wnt pathway components. Immunofluorescence studies can visualize β-catenin nuclear translocation in response to ADGRA2-mediated signaling. For more comprehensive analyses, combine these approaches with RNA-seq or proteomics to identify downstream effectors and feedback mechanisms in the signaling cascade.

How should I interpret discrepancies in ADGRA2 detection between different antibodies?

Discrepancies in ADGRA2 detection between antibodies may arise from several factors. Different epitopes may be differentially accessible due to protein conformation, post-translational modifications, or protein-protein interactions. Some antibodies may recognize specific isoforms or processed forms of ADGRA2. To address discrepancies, map the epitopes recognized by each antibody, verify results with multiple antibodies targeting different regions, and correlate protein detection with mRNA expression data. Consider the possibility that different antibodies may be revealing biologically relevant states or forms of the protein.

What methods can validate the specificity of ADGRA2 antibody staining?

Multiple approaches can validate antibody specificity. First, genetic approaches using CRISPR/Cas9-mediated knockout cells or tissues provide definitive negative controls . Second, RNA interference (siRNA or shRNA) to knockdown ADGRA2 expression should reduce antibody signal proportionally to knockdown efficiency. Third, pre-adsorption with the immunizing peptide should eliminate specific staining. Fourth, correlation of staining patterns with mRNA expression (by in situ hybridization or single-cell RNA-seq) can provide additional validation. Finally, comparison of staining patterns across multiple antibodies targeting different epitopes can confirm specificity.

How can I quantitatively analyze ADGRA2 expression across different experimental conditions?

For Western blot quantification, use internal loading controls (such as GAPDH, β-actin, or tubulin) and densitometry software to normalize ADGRA2 signals. For immunofluorescence quantification, define appropriate parameters (mean fluorescence intensity, area of positive staining, or subcellular distribution patterns) and analyze multiple fields and biological replicates. Flow cytometry can provide population-level quantification of surface-expressed ADGRA2. For absolute quantification, consider developing a standard curve using recombinant ADGRA2 protein of known concentration.

What considerations apply when studying ADGRA2 in patient-derived tissues?

When working with patient-derived tissues, several factors require attention. First, ensure appropriate ethical approvals and informed consent. Second, account for potential genetic variations that might affect antibody binding, particularly if the antibody targets a region with known polymorphisms. Third, consider fixation artifacts and optimize antigen retrieval methods for archived tissues. Fourth, select appropriate normal controls from similar demographic backgrounds. Finally, correlate protein expression with clinical data to establish meaningful relationships while maintaining rigorous blinding procedures during analysis.

How can ADGRA2 antibodies be used to investigate developmental processes?

ADGRA2 plays crucial roles in cerebrovascular development . For developmental studies, timing is critical—establish precise developmental staging and use antibodies compatible with embryonic tissues. Section thickness and clearing techniques may need optimization for deep-tissue imaging. Consider co-staining with stage-specific markers to contextualize ADGRA2 expression patterns. For functional studies, combine antibody detection with genetic approaches (such as conditional knockout models) to correlate protein expression with developmental phenotypes. Time-course analyses can reveal dynamic changes in expression and localization during development.

What approaches can address the challenges of studying ADGRA2 in highly vascularized tissues?

Since ADGRA2 is expressed in endothelial cells , studying it in vascularized tissues presents challenges in distinguishing specific signals from background. Dual immunofluorescence with endothelial markers (CD31, VE-cadherin) can help identify ADGRA2-positive vessels. Laser capture microdissection may isolate specific vascular beds for protein extraction and analysis. Tissue clearing techniques combined with confocal or light-sheet microscopy can provide three-dimensional visualization of ADGRA2 expression in intact vascular networks. Single-cell approaches may help delineate heterogeneity in ADGRA2 expression across different endothelial subpopulations.