ADORA2B Antibody

What is ADORA2B and why is it important in research?

ADORA2B (Adenosine A2b receptor) is a member of the G protein-coupled receptor superfamily that functions as an integral membrane protein. It stimulates adenylate cyclase activity in the presence of adenosine and interacts with netrin-1, which is involved in axon elongation. The gene is located near the Smith-Magenis syndrome region on chromosome 17. ADORA2B has gained significant research interest due to its involvement in various physiological and pathological processes, particularly in cancer progression and metastasis. The receptor has been found to be overexpressed in multiple cancer types, including gastric cancer, lung adenocarcinoma, breast cancer, and head and neck squamous cell carcinoma, making it an important target for cancer research .

What are the common applications for ADORA2B antibodies in research?

ADORA2B antibodies are utilized in various research applications to detect and study the receptor's expression, localization, and function. The primary applications include Western Blot (WB) for protein detection and quantification, Immunofluorescence (IF) for cellular localization studies, Immunohistochemistry (IHC) for tissue expression analysis, Flow Cytometry (FCM) for cellular expression analysis, and ELISA for quantitative detection. These techniques allow researchers to investigate ADORA2B expression patterns in different tissues, cell types, and disease states. For instance, commercially available antibodies like Proteintech's 21071-1-AP and Bioss's bs-10205R have been validated for these applications with specific dilution recommendations for optimal results .

How should researchers select the appropriate ADORA2B antibody for their specific experiment?

When selecting an ADORA2B antibody, researchers should consider several factors to ensure experimental success. First, verify the antibody's reactivity with your species of interest; commercially available antibodies show reactivity with human, mouse, and rat samples. Second, confirm the antibody has been validated for your specific application (WB, IF, IHC, etc.) by reviewing validation data and publications. Third, consider the antibody type (polyclonal vs. monoclonal) based on your research needs; polyclonal antibodies like 21071-1-AP from Proteintech offer high sensitivity but potentially lower specificity compared to monoclonals. Fourth, examine the immunogen information to understand the antibody's epitope recognition, which is crucial for detecting specific isoforms or regions of ADORA2B. Finally, review published literature using these antibodies to gauge their performance in experimental conditions similar to yours .

How does ADORA2B signaling affect epithelial-mesenchymal transition (EMT) in cancer progression?

ADORA2B has been demonstrated to play a significant role in regulating epithelial-mesenchymal transition (EMT), which is crucial for cancer metastasis. Research focused on gastric cancer (GC) has revealed that ADORA2B significantly influences the expression of EMT marker proteins and consequently regulates the invasion and migration capabilities of GC cells. Specifically, studies have shown that ADORA2B expression levels are significantly higher in GC tissue, metastatic greater omental tissue, and lymphatic metastasis tissue compared to paracancerous tissue, correlating with lymph node metastasis and invasion. ADORA2B appears to modulate the expression of key EMT markers including β-catenin, N-cadherin, vimentin, and E-cadherin. The mechanistic relationship involves ADORA2B-mediated regulation of these proteins, promoting a more invasive cellular phenotype. Interestingly, antagonizing ADORA2B expression through specific inhibitors like PSB-603 can reverse EMT marker protein expression, suggesting a potential therapeutic approach for limiting cancer metastasis .

What is the relationship between ADORA2B and tumor immunity, and how can researchers investigate this connection?

ADORA2B plays a complex role in tumor immunology, with significant implications for cancer immunotherapy research. Studies have demonstrated that inhibition of adenosine-producing enzymes and adenosine A2A or A2B receptors can stimulate antitumor immunity and limit tumor progression. Specifically, inhibition of ADORA2B has been shown to significantly enhance antitumor immune responses. To investigate this connection, researchers can employ several approaches: (1) Use ADORA2B antagonists (such as PSB-603) in combination with immune checkpoint inhibitors in tumor models to assess synergistic effects; (2) Analyze immune cell infiltration and activation in tumors after ADORA2B inhibition using flow cytometry and immunohistochemistry; (3) Evaluate changes in cytokine production and T-cell function in the tumor microenvironment following ADORA2B modulation; (4) Compare ADORA2B expression in tumor tissues with the degree of immune cell infiltration using immunohistochemical staining with antibodies targeting both ADORA2B and immune cell markers; (5) Perform co-culture experiments with tumor cells and immune cells in the presence of ADORA2B modulators to directly assess immune activation and tumor cell killing. These approaches can help elucidate the mechanistic basis of ADORA2B's role in tumor immunity .

How can researchers effectively study the combined effects of ADORA2B antagonists with chemotherapeutic agents?

To effectively study the combined effects of ADORA2B antagonists with chemotherapeutic agents, researchers should implement a multi-faceted experimental approach. Based on studies examining the combination of ADORA2B antagonist (PSB-603) with cisplatin in gastric cancer, the following methodology is recommended: First, establish appropriate cell culture models expressing ADORA2B, which can be verified using validated antibodies through Western blot or immunofluorescence. Second, perform dose-response studies with both the ADORA2B antagonist and chemotherapeutic agent separately to determine effective concentrations. Third, design combination treatment experiments using different treatment schedules (concurrent, sequential) and concentration ratios to identify optimal combinations. Fourth, assess multiple endpoints including cell viability (MTT/XTT assays), apoptosis (flow cytometry with Annexin V/PI staining), cell cycle analysis, and invasion/migration assays (transwell/wound healing). Fifth, examine molecular changes in EMT markers through protein expression analysis via Western blotting using antibodies against ADORA2B, E-cadherin, N-cadherin, vimentin, and β-catenin. Finally, validate findings in animal models to assess tumor growth inhibition, metastasis prevention, and survival improvement. This comprehensive approach will provide insights into how ADORA2B inhibition might sensitize cancer cells to chemotherapy, potentially revealing new therapeutic strategies .

What are the optimal protocols for using ADORA2B antibodies in Western blotting experiments?

For optimal Western blotting with ADORA2B antibodies, researchers should follow a systematic protocol while considering antibody-specific parameters. Begin with sample preparation from relevant tissues (mouse brain tissue has been validated as a positive control) or cell lines, using a complete lysis buffer containing protease inhibitors to preserve protein integrity. For protein separation, use 10-12% SDS-PAGE gels to effectively resolve the 36 kDa ADORA2B protein. After transferring proteins to a PVDF or nitrocellulose membrane, block with 5% non-fat milk or BSA in TBST for 1-2 hours at room temperature. For primary antibody incubation, dilute the ADORA2B antibody according to manufacturer recommendations: Proteintech's 21071-1-AP at 1:200-1:1000, Bioss's bs-10205R at 1:300-5000, or other validated antibodies at their specified concentrations. Incubate overnight at 4°C for optimal binding. After washing with TBST (3-5 times for 5-10 minutes each), apply appropriate HRP-conjugated secondary antibody (typically anti-rabbit IgG for the polyclonal antibodies listed) at 1:2000-1:5000 dilution and incubate for 1-2 hours at room temperature. Following additional washing steps, develop using ECL reagent and image with appropriate detection equipment. When interpreting results, expect bands at approximately 36 kDa, though variations may occur due to post-translational modifications or different isoforms .

How should researchers optimize immunohistochemistry protocols for ADORA2B detection in tissue samples?

Optimizing immunohistochemistry (IHC) protocols for ADORA2B detection requires careful attention to several critical steps. Begin with proper tissue fixation, typically using 10% neutral buffered formalin, followed by paraffin embedding and sectioning at 4-6 μm thickness. For antigen retrieval, which is essential for most formalin-fixed tissues, use citrate buffer (pH 6.0) or EDTA buffer (pH 9.0) with heat-induced epitope retrieval methods (pressure cooker or microwave) to expose masked epitopes. Block endogenous peroxidase activity with 3% hydrogen peroxide for 10-15 minutes, followed by protein blocking with 5-10% normal serum (matching the species of the secondary antibody) to reduce non-specific binding. For primary antibody incubation, dilute ADORA2B antibodies according to manufacturer recommendations: Bioss's bs-10205R at 1:200-400 for IHC-P, or Boster Bio's A05238 at the recommended dilution. Incubate overnight at 4°C in a humidified chamber for optimal results. Use an appropriate detection system (such as HRP-polymer or biotin-streptavidin) and visualize with DAB chromogen. Counterstain with hematoxylin to provide tissue context. Include positive controls (tissues known to express ADORA2B, such as certain cancer tissues) and negative controls (primary antibody omission) in each experiment. For dual staining with other markers (such as EMT markers), follow sequential staining protocols with appropriate chromogens or fluorescent secondaries if performing immunofluorescence .

What controls and validation steps are essential when working with ADORA2B antibodies in research?

Implementing comprehensive controls and validation steps is crucial when working with ADORA2B antibodies to ensure experimental reliability and data integrity. First, include positive control samples with known ADORA2B expression; mouse brain tissue has been validated for Western blotting, while human gastric cancer, lung adenocarcinoma, or breast cancer tissues are appropriate for immunohistochemistry based on literature findings. Second, incorporate negative controls including tissues or cells with minimal ADORA2B expression and technical controls where primary antibody is omitted or replaced with non-specific IgG from the same species. Third, validate antibody specificity through multiple approaches: (a) peptide competition assays where pre-incubation of the antibody with its immunizing peptide should abolish specific staining; (b) siRNA knockdown or CRISPR knockout of ADORA2B in cell lines followed by antibody testing; (c) comparison of staining patterns across multiple ADORA2B antibodies targeting different epitopes. Fourth, confirm subcellular localization consistency with known ADORA2B biology (primarily cell membrane localization). Fifth, perform Western blotting to verify the correct molecular weight (approximately 36 kDa, though posttranslational modifications may alter the observed weight). Finally, cross-reference your findings with published literature to ensure consistency with established ADORA2B expression patterns and functions in your experimental system .

How can researchers effectively use ADORA2B antibodies in co-localization studies with other proteins?

To effectively conduct co-localization studies of ADORA2B with other proteins, researchers should implement a methodical approach combining proper experimental design with rigorous imaging techniques. Begin by selecting compatible primary antibodies raised in different host species (e.g., rabbit anti-ADORA2B combined with mouse antibodies against potential interacting partners) to allow for distinct secondary antibody detection. For immunofluorescence applications, optimize the signal-to-noise ratio for each antibody individually before performing co-staining, adjusting dilutions and incubation conditions as needed. When performing the co-staining protocol, apply primary antibodies either sequentially (for more challenging samples) or simultaneously (if cross-reactivity isn't an issue), followed by fluorophore-conjugated secondary antibodies with non-overlapping emission spectra (typically Alexa Fluor 488 for ADORA2B as documented in gastric cancer studies, paired with Alexa Fluor 594/647 for partner proteins). Include appropriate controls: single-antibody stainings to rule out bleed-through, controls omitting each primary antibody to confirm secondary antibody specificity, and positive controls where co-localization is expected or known. For imaging, use confocal microscopy with sequential scanning to minimize cross-channel contamination. Analyze co-localization quantitatively using coefficients such as Pearson's or Mander's, and complement this with biochemical approaches (co-immunoprecipitation) to confirm physical interactions suggested by co-localization. This approach is particularly valuable for studying relationships between ADORA2B and EMT markers or components of adenylate cyclase signaling pathways .

How can researchers address non-specific binding or high background issues when using ADORA2B antibodies?

Non-specific binding and high background are common challenges when working with ADORA2B antibodies that can be systematically addressed through protocol optimization. For Western blotting applications, increase blocking stringency by extending blocking time to 2 hours or overnight at 4°C using 5% BSA instead of milk, particularly if phospho-specific detection is involved. Enhance washing efficiency with longer and more frequent TBST washes (5-6 times for 10 minutes each). Optimize primary antibody concentration by performing titration experiments; for instance, with Proteintech's 21071-1-AP, test across the recommended 1:200-1:1000 range to identify the optimal concentration that maximizes specific signal while minimizing background. For immunohistochemistry and immunofluorescence, improve tissue preparation through extended fixation time optimization, thorough deparaffinization, and effective permeabilization. Consider adding 0.1-0.3% Triton X-100 to blocking solutions to reduce non-specific membrane interactions. Implement additional blocking steps with 10% serum from the secondary antibody's host species and include 1% BSA in antibody diluents. For particularly challenging samples, consider using commercially available background-reducing reagents or try various antigen retrieval methods (heat-induced versus enzymatic). Finally, ensure secondary antibodies are highly cross-adsorbed against the species being examined and are used at appropriate dilutions (typically 1:500-1:2000). When troubleshooting, modify one parameter at a time while maintaining detailed records of protocol changes and their effects on signal-to-noise ratio .

What strategies should be employed when ADORA2B antibodies fail to detect the protein in experimental samples?

When ADORA2B antibodies fail to detect the protein in experimental samples, researchers should implement a systematic troubleshooting approach addressing sample preparation, detection conditions, and antibody selection. First, verify ADORA2B expression in your experimental system through RT-qPCR to confirm mRNA presence before attempting protein detection. Second, optimize protein extraction methods; for membrane-bound proteins like ADORA2B, ensure your lysis buffer contains appropriate detergents (0.5-1% NP-40, Triton X-100, or CHAPS) and consider using specialized membrane protein extraction kits. Third, protect the protein from degradation by including fresh protease inhibitors in lysis buffers and maintaining samples at cold temperatures during processing. Fourth, for Western blotting, adjust protein loading (increase to 50-80 μg for low-abundance proteins), optimize transfer conditions (consider longer transfer times or semi-dry systems for membrane proteins), and explore various blocking agents (BSA may be preferable to milk for certain epitopes). Fifth, test different antibody concentrations beyond the manufacturer's recommended range and extend incubation times (overnight at 4°C). Sixth, consider epitope accessibility issues by trying multiple ADORA2B antibodies targeting different regions of the protein; for instance, compare results between Proteintech's 21071-1-AP and Bioss's bs-10205R which recognize different epitopes. Finally, include appropriate positive controls (mouse brain tissue has been validated for ADORA2B detection) in each experiment. If these approaches fail, consider alternative detection methods such as immunoprecipitation followed by Western blotting, which can enrich for low-abundance proteins .

What are the best approaches for quantifying ADORA2B expression levels in different experimental models?

Quantifying ADORA2B expression levels accurately across different experimental models requires a multi-technique approach that accounts for the protein's characteristics and experimental context. For relative protein quantification, Western blotting offers a reliable method when performed with proper controls. Optimize sample loading (30-50 μg total protein), ensure equal loading through housekeeping protein normalization (β-actin, GAPDH, or α-tubulin), and use digital imaging systems for densitometric analysis. For cell-by-cell expression analysis, flow cytometry provides quantitative data on ADORA2B surface expression levels; antibodies like Bioss's bs-10205R have been validated for FCM applications at 1:20-100 dilutions. For tissue expression analysis, quantitative immunohistochemistry can be performed using digital image analysis software to measure staining intensity and distribution after standardizing staining conditions across all samples. For absolute quantification, develop an ELISA system using validated ADORA2B antibodies as capture and detection antibodies, creating standard curves with recombinant ADORA2B protein. At the mRNA level, RT-qPCR offers a complementary approach to verify protein data, though correlation between mRNA and protein levels should not be assumed without verification. For spatial expression patterns, quantitative immunofluorescence with confocal microscopy allows for subcellular localization analysis. When comparing results across different experimental models (cell lines, primary cultures, tissue samples), it's essential to maintain consistent protocols, use the same antibody lots, and include appropriate positive controls (such as mouse brain tissue for ADORA2B detection) in each experiment. This comprehensive approach provides robust quantitative data on ADORA2B expression across experimental systems .