ADORA1 Antibody

What is the ADORA1 receptor and why is it significant in research?

ADORA1 (adenosine receptor A1) is a member of the 7-transmembrane domain G-protein-coupled receptor superfamily that mediates several critical physiological processes including cardiac rate regulation, smooth muscle tone, platelet aggregation, inflammation, cell growth/death, and neurotransmission . Its significance lies in its widespread expression and diverse functions across multiple systems, making it a valuable target for both basic research and therapeutic development. The receptor is highly expressed in brain tissues (especially cerebellum, hippocampus, thalamus, and cortex), spinal cord, and also appears in kidney, testis, and white adipocytes where it inhibits lipolysis and stimulates glucose uptake .

How do I select the appropriate ADORA1 antibody for my specific application?

Selection depends primarily on your experimental application, species of interest, and detection requirements. For Western blot applications in rat tissues, polyclonal antibodies like PA1-041A detect an ~37 kDa protein in rat brain samples . For human samples, monoclonal antibodies such as clone 10D12 (ab288377) have been validated for Western blot and IHC-P applications . When working with mouse samples, consider PA3-041, which demonstrates stronger reactivity in mouse than human tissues . Cross-reactivity should be carefully evaluated - for instance, PA1-041A specifically detects A1AR without cross-reacting with other adenosine receptor subtypes . Always review validation data for your specific application before selection.

What are the molecular mechanisms through which ADORA1 mediates its cellular effects?

At the molecular level, ADORA1 is a glycoprotein that activates Gi and Go proteins in vitro . Upon agonist occupation, ADORA1 causes pertussis toxin-sensitive inhibition of adenylyl cyclase activity, reducing intracellular cAMP levels . In some cellular systems, ADORA1 activation stimulates phospholipase C, resulting in mobilization of intracellular calcium stores . Additionally, ADORA1 activates potassium channels, which contributes significantly to its pronounced effects on the cardiovascular system . In white adipocytes, ADORA1 activation inhibits lipolysis and stimulates glucose uptake through these signaling pathways . These mechanisms collectively explain how ADORA1 can produce diverse physiological responses across different tissue types.

What are the optimal protocols for ADORA1 antibody application in immunohistochemistry?

For paraffin-embedded tissues, use validated antibodies like ab288377 (clone 10D12) at a 1:100 dilution . After dewaxing and hydration, conduct antigen retrieval under high pressure in citrate buffer (pH 6.0) . For frozen sections, antibodies like AAR-006 have been validated for rat cortex and medial septum . When dual-labeling, ADORA1 detection pairs effectively with neuronal markers like parvalbumin to identify receptor expression in specific neuronal subtypes . Visualization typically employs fluorophore-conjugated secondary antibodies (e.g., goat anti-rabbit AlexaFluor-555) . For optimal results, include both positive controls (tissues with known high expression, such as cerebellum) and negative controls (primary antibody omission or peptide competition controls). Always validate specificity with blocking peptides where available .

How should I optimize Western blot protocols for detecting ADORA1?

For optimal Western blot detection of ADORA1, prepare samples in denaturing conditions with complete protease inhibitors to prevent degradation. For brain tissue lysates, polyclonal antibodies like PA1-041A detect ADORA1 at ~37 kDa . For human cell lines, monoclonal antibody ab288377 (clone 10D12) has been validated at 1/2000 dilution on A549, HEK-293, and U-87 MG cell lysates . After SDS-PAGE separation and transfer, block membranes with 5% non-fat milk or BSA in TBST. Primary antibody incubation typically requires overnight incubation at 4°C, followed by species-appropriate HRP-conjugated secondary antibodies. When troubleshooting, validate specificity through blocking peptides, as demonstrated with AAR-006 where peptide pre-incubation eliminates the specific band . For kidney samples, which show lower expression, longer exposure times may be necessary compared to brain samples .

What considerations are important for immunocytochemistry applications of ADORA1 antibodies?

For immunocytochemistry, fixation methodology significantly impacts antibody performance. Paraformaldehyde fixation (4%, 15-20 minutes) followed by 0.1% Triton X-100 permeabilization is effective for ADORA1 detection in primary cultures such as dorsal root ganglion (DRG) cells . When working with neuronal cultures, antibodies like AAR-006 can be used at 1:100 dilution . For cellular localization studies, combine ADORA1 staining with membrane markers to confirm receptor placement at the cell surface, and nuclear counterstains like Hoechst 33342 to provide cellular context . During imaging, pay special attention to receptor internalization under various treatment conditions, as GPCR trafficking can provide insights into receptor function and regulation. When examining co-localization with other proteins, confocal microscopy is recommended for accurate spatial resolution.

How can I address non-specific binding issues with ADORA1 antibodies?

Non-specific binding with ADORA1 antibodies can be addressed through several methodological approaches. First, validate antibody specificity using blocking peptides where available – preincubating antibodies like AAR-006 with their specific blocking peptide (BLP-AR006) should eliminate specific signal in control experiments . Optimize antibody concentration through titration; for Western blot, dilutions ranging from 1:500 to 1:2000 are typically effective . For immunohistochemistry applications, 1:50 to 1:200 dilutions are commonly used . Adjust blocking conditions using 5-10% serum from the secondary antibody host species or 3-5% BSA to reduce background. Consider using highly cross-adsorbed secondary antibodies to minimize cross-reactivity. Importantly, know that PA1-041A specifically recognizes A1AR without detecting other adenosine receptor subtypes, making it valuable for discriminating between receptor isoforms .

How can I interpret contradictory results between different ADORA1 antibodies?

Contradictory results between different ADORA1 antibodies often stem from epitope differences and varying specificities. Analyze the immunogen information: PA1-041A targets synthetic peptide C(309)QPKPPIDEDLPEEEKAED(326) of rat ADORA1 , while PA3-041 uses a mix of peptides corresponding to C-terminal portions of human C(309)QPAPPIDEDLPEERPDDD(326) and mouse C(309)QPKPPIEEDIPEEEKADD(326) sequences . Epitope accessibility may differ in various experimental conditions or tissue preparations. Verify results using multiple antibodies targeting different ADORA1 epitopes. For definitive validation, consider knockout/knockdown controls or heterologous expression systems with tagged receptors. Species differences can be significant – PA3-041 shows stronger reactivity in mouse than human samples and is only recommended for overexpressed or recombinant human samples . When contradictions persist, functional assays measuring ADORA1 activity may help resolve discrepancies.

What are the critical variables affecting ADORA1 detection in different tissue types?

Detection of ADORA1 across different tissues requires consideration of multiple critical variables. Expression levels vary significantly – ADORA1 is highly expressed in brain (especially cerebellum, hippocampus, thalamus, and cortex) but shows lower expression in kidney and testis . For low-expressing tissues, signal amplification techniques (like TSA) may be necessary. Sample preparation significantly impacts results: perfusion-fixed tissues typically yield better results than immersion-fixed samples for brain tissues. Post-mortem interval critically affects receptor integrity, particularly for human samples. Fixation parameters (duration, temperature, pH) must be optimized for each tissue type. For Western blot applications, lipid-rich tissues may require additional extraction steps to effectively solubilize this membrane-bound receptor. Receptor phosphorylation states and internalization dynamics may differ between tissues, affecting epitope accessibility and antibody binding. Consider tissue-specific expression of ADORA1 splice variants when interpreting band patterns.

How can ADORA1 antibodies be utilized in receptor trafficking and internalization studies?

ADORA1 antibodies can be strategically employed to investigate receptor trafficking through multiple methodologies. For real-time tracking, combine surface-labeling of live cells using non-permeabilizing conditions with subsequent internalization assays. After agonist stimulation (e.g., with adenosine or selective A1 agonists), quantify surface receptor loss using flow cytometry with antibodies like ACDG-1 . For microscopy approaches, use immunofluorescence with AAR-006 to visualize receptor translocation from membrane to intracellular compartments . Co-localization with endosomal markers (Rab5, Rab7, Rab11) can distinguish between degradation and recycling pathways. For biochemical quantification, employ surface biotinylation followed by Western blot with PA1-041A or similar antibodies . Advanced approaches include antibody feeding assays where surface receptors are labeled with primary antibody before internalization is triggered, allowing selective visualization of the internalized receptor pool. ADORA1 trafficking studies are particularly relevant in neurons where receptor redistribution may influence synaptic transmission.

What approaches can be used to study ADORA1 heteromerization with other GPCRs?

Studying ADORA1 heteromerization with other GPCRs requires sophisticated methodological approaches. Proximity ligation assays (PLA) using pairs of antibodies against ADORA1 (such as PA1-041A) and potential partner receptors can detect protein-protein interactions within 40nm in fixed tissues or cells . For co-immunoprecipitation studies, use antibodies like ACDG-1 with verified IP capability (1:50 dilution) to pull down ADORA1 complexes and probe for interacting partners . FRET/BRET approaches using antibody-based fluorophore conjugates can measure energy transfer between closely associated receptors. Super-resolution microscopy (STORM, PALM) combined with multi-color immunolabeling using antibodies like AAR-006 enables visualization of receptor clusters at nanoscale resolution . When analyzing potential heteromers, consider tissue-specific expression patterns - ADORA1 is highly expressed in brain regions like cerebellum, hippocampus, thalamus, and cortex, making these valuable regions for heteromer investigation . Control experiments should include competitive displacement with receptor-specific ligands to confirm specificity of interactions.

How can phospho-specific antibodies enhance our understanding of ADORA1 signaling dynamics?

While the provided search results don't specifically reference phospho-specific ADORA1 antibodies, this approach represents an advanced frontier in receptor research. Phosphorylation of ADORA1 at specific residues modulates its activity, desensitization, and interaction with signaling partners. Researchers would benefit from developing and validating phospho-specific antibodies targeting key regulatory sites, particularly those phosphorylated by GRKs (G protein-coupled receptor kinases), PKA, and PKC. Such antibodies could be used in Western blot analysis to track temporal dynamics of receptor phosphorylation following agonist stimulation. In immunohistochemistry applications, phospho-specific antibodies could reveal differential phosphorylation states across brain regions where ADORA1 is highly expressed, such as cerebellum, hippocampus, thalamus, and cortex . For signaling studies, multiplexed detection of phosphorylated ADORA1 alongside downstream effectors (phospho-ERK, phospho-CREB) would provide integrated pathway analysis. Researchers should validate phospho-specific antibodies using phosphatase treatments and phospho-null receptor mutants.

What are the key differences in ADORA1 detection across species?

Significant species variations exist in ADORA1 detection that researchers must consider. PA1-041A is optimized for rat tissues and does not detect other AR subtypes, but previous lots have worked with human and bovine samples . PA3-041 detects both human and mouse ADORA1, but shows stronger reactivity in mouse and is only recommended for overexpressed or recombinant human samples . The epitope differences are evident in immunizing sequences: for human ADORA1, C(309)QPAPPIDEDLPEERPDDD(326), while for mouse, C(309)QPKPPIEEDIPEEEKADD(326) . Monoclonal antibody ab288377 (clone 10D12) has been validated specifically for human samples in multiple cell lines (A549, HEK-293, U-87 MG) . For rat brain sections, AAR-006 effectively labels neurons and astrocytes . These species differences likely reflect variations in amino acid sequences at antibody binding sites, particularly in the C-terminal regions that serve as common immunogens. When transitioning between species, preliminary validation experiments are essential before proceeding to full studies.

How should I approach epitope mapping for ADORA1 antibodies in different model systems?

When approaching epitope mapping for ADORA1 antibodies across different model systems, begin by analyzing sequence alignments of the target regions. The C-terminal portion shows species variations: human ADORA1 sequence is C(309)QPAPPIDEDLPEERPDDD(326), while rat/mouse sequences are C(309)QPKPPIDEDLPEEEKAED(326) and C(309)QPKPPIEEDIPEEEKADD(326), respectively . For experimental epitope mapping, generate a series of deletion constructs or peptide fragments spanning the presumed epitope region for competition assays. Test antibody binding to these fragments using ELISA or dot blot approaches. Alternatively, employ alanine scanning mutagenesis of key residues within the epitope region to identify critical binding determinants. For comprehensive mapping, hydrogen-deuterium exchange mass spectrometry (HDX-MS) can identify specific interaction sites between antibody and antigen. When working with conformational epitopes, use native gel electrophoresis and non-denaturing conditions to preserve epitope structure. Cross-species reactivity predictions should be validated experimentally, particularly when sequence homology is below 90%.

What methodological adaptations are required when transitioning from rodent to human ADORA1 research?

Transitioning from rodent to human ADORA1 research requires several critical methodological adaptations. Antibody selection is paramount - while PA1-041A works primarily with rat samples, researchers working with human tissues should select antibodies like clone 10D12 (ab288377) specifically validated for human samples . PA3-041 detects both human and mouse ADORA1 but shows stronger reactivity in mouse and is recommended only for overexpressed or recombinant human samples . For immunohistochemistry on human brain tissues, optimize antigen retrieval conditions (high-pressure citrate buffer, pH 6.0) . Human tissues typically require longer fixation times and may benefit from alternative antigen retrieval methods compared to rodent samples. Be aware that post-mortem interval significantly impacts receptor integrity in human tissues. When analyzing Western blot data, note that human ADORA1 typically appears at 37 kDa, similar to rodent samples . For functional studies, remember that pharmacological profiles may differ between species; human ADORA1 may show different agonist/antagonist potency and kinetics compared to rodent receptors.