adra2a Antibody

What is ADRA2A and why is it a significant research target?

ADRA2A (adrenergic, alpha-2A-, receptor) belongs to the adrenoceptor alpha-2 family of GPCRs coupled to the inhibitory G-alpha subunit (Gi). It functions primarily by suppressing adenylyl cyclase activity, which inhibits the cyclization of AMP to cAMP . ADRA2A plays a crucial role in the central nervous system by inhibiting sympathetic nerve flow, contributing to sedation, anxiolysis, and antinociception . Recent evidence also suggests its importance in pancreatic cancer, where reduced ADRA2A expression correlates with the aggressive basal-like/squamous subtype, increased lymph node metastasis, higher pathological grade, advanced disease stage, and decreased patient survival . These diverse functions make ADRA2A an important target for neuroscience, pharmacology, and oncology research.

What are the primary applications for ADRA2A antibodies in research?

ADRA2A antibodies have multiple validated research applications. Based on published literature, these antibodies are primarily used for:

These applications enable researchers to investigate ADRA2A expression, localization, and function across various experimental contexts . The antibody's versatility allows for comprehensive characterization of ADRA2A in different biological systems and disease models.

What species reactivity can be expected with commercially available ADRA2A antibodies?

Most commercially available ADRA2A antibodies demonstrate cross-reactivity with multiple species. According to the search results, several antibodies show tested reactivity with human, mouse, and rat samples . This multi-species reactivity is particularly valuable for comparative studies and translational research. For instance, antibody 14266-1-AP has been validated for all three species in Western Blot applications . When selecting an antibody for your specific experimental system, it's advisable to choose one with documented reactivity in your species of interest, preferably with validation data in your specific application context.

How can ADRA2A antibodies be used to investigate receptor signaling and disease mechanisms?

ADRA2A antibodies serve as powerful tools for elucidating receptor signaling pathways and disease mechanisms. In pancreatic cancer research, ADRA2A antibodies have been instrumental in demonstrating that ADRA2A suppresses the aggressive basal-like/squamous subtype . Methodologically, researchers have utilized these antibodies in transcriptome and metabolome analyses to identify ADRA2A's role in molecular subtype differentiation and its influence on metabolic features .

For signaling studies, ADRA2A antibodies can be used to:

• Quantify receptor expression levels via Western blotting

• Visualize receptor localization through immunohistochemistry and immunofluorescence

• Investigate protein-protein interactions via co-immunoprecipitation

• Monitor receptor trafficking and internalization using live-cell imaging combined with IF

In disease models, researchers have employed immunohistochemistry with ADRA2A antibodies (1:200 dilution) on paraffin-embedded sections to correlate expression with clinical outcomes, assigning intensity scores (0-3) and prevalence scores (0-4) to generate comprehensive IHC scores . This approach provides quantitative data for statistical analyses linking ADRA2A expression to disease progression.

What considerations should be made when designing ADRA2A co-localization or interaction studies?

When designing co-localization or interaction studies involving ADRA2A, several methodological considerations are critical. Evidence suggests ADRA2A can interact with other receptors, such as TAAR1, as demonstrated through co-immunoprecipitation and ELISA-based methods . For optimal experimental design:

• Antibody selection: Choose antibodies targeting different epitopes to avoid steric hindrance. The search results identify multiple antibodies targeting different regions of ADRA2A (AA 240-380, AA 385-450, AA 7-20, etc.) , allowing for strategic selection based on the experimental question.

• Controls: Include both positive and negative controls. For instance, the growth hormone secretagogue receptor (GHSR) homo-oligomer has been used as a positive control in ADRA2A-TAAR1 interaction studies .

• Expression systems: Consider using established cell lines like COS-7 cells for co-expression studies, which provide robustness through multiple experimental manipulations .

• Tagged constructs: Utilize differentially tagged constructs (e.g., N-terminally HA-tagged ADRA2A and C-terminally Flag-tagged interaction partners) to facilitate detection of protein complexes .

• Validation approaches: Combine multiple methodologies (co-immunoprecipitation, proximity ligation assays, FRET/BRET) to confirm interactions with confidence.

These methodological considerations ensure rigorous investigation of ADRA2A's role in protein complexes and signaling networks.

How can expression patterns of ADRA2A be accurately quantified across different tissues or disease states?

Accurate quantification of ADRA2A expression across tissues or disease states requires a multi-modal approach. Based on the research methods described in the search results, the following methodological framework is recommended:

This comprehensive approach enables robust quantification of ADRA2A expression differences, particularly important in comparative studies between normal and pathological states.

What are the optimal protocols for ADRA2A antibody use in Western blotting applications?

For optimal Western blotting with ADRA2A antibodies, the following methodology is recommended based on published protocols:

• Sample preparation:

  • PC-12 cells have been validated as positive controls for ADRA2A detection

  • Maintain complete protease inhibitor cocktail in lysis buffers

  • For tissues, flash-freeze immediately and process with appropriate homogenization

• Protein loading and separation:

  • Load 20-50 μg of total protein per lane

  • Use 8-12% SDS-PAGE gels for optimal separation

  • Include molecular weight markers to verify the observed molecular weight (approximately 49-51 kDa)

• Transfer and blocking:

  • Transfer to PVDF or nitrocellulose membranes

  • Block with 5% non-fat milk or BSA in TBST

• Primary antibody incubation:

  • Use ADRA2A antibodies at dilutions between 1:200-1:1000

  • Incubate overnight at 4°C for optimal signal-to-noise ratio

  • Consider the specific epitope recognition characteristics of your selected antibody

• Detection and analysis:

  • Use appropriate HRP-conjugated secondary antibodies

  • Include positive controls (PC-12 cell lysates) and negative controls

  • Be aware that ADRA2A has a calculated molecular weight of 49 kDa but may be observed at approximately 51 kDa due to post-translational modifications

This protocol has been validated in multiple publications and provides reliable detection of ADRA2A protein in various sample types.

What are the critical considerations for optimizing immunohistochemistry protocols with ADRA2A antibodies?

Successful immunohistochemistry with ADRA2A antibodies requires careful optimization of several critical parameters:

• Tissue preparation and fixation:

  • Paraffin-embedded sections of 4 μm thickness are recommended

  • Proper fixation (10% neutral buffered formalin) is crucial for epitope preservation

• Antigen retrieval:

  • TE buffer pH 9.0 is suggested as the primary antigen retrieval method

  • Alternatively, citrate buffer pH 6.0 can be used if the primary method yields suboptimal results

  • Heat-induced epitope retrieval (pressure cooker or microwave) improves antibody accessibility

• Antibody concentration and incubation:

  • Dilute ADRA2A antibodies between 1:200-1:800

  • Overnight incubation at 4°C is recommended for optimal binding

  • Consider using a humidity chamber to prevent section drying

• Detection system:

  • The Dako envision + system-HRP labeled polymer anti-rabbit antibody has been successfully employed

  • Develop with 3,3'-diaminobenzidine (DAB) for visualization

• Controls and validation:

  • Include positive controls (mouse brain tissue has been validated)

  • Include negative controls (omitting primary antibody)

  • For quantification, employ standardized scoring systems based on intensity (0-3) and prevalence (0-4) metrics

These methodological considerations ensure reproducible and specific detection of ADRA2A in tissue sections, enabling reliable comparative studies across different experimental conditions.

How can specificity of ADRA2A antibodies be validated in experimental systems?

Validating antibody specificity is crucial for ensuring reliable research outcomes. For ADRA2A antibodies, a comprehensive validation strategy should include:

• Genetic approaches:

  • Test antibody reactivity in ADRA2A knockout or knockdown models

  • Compare signal in cells with endogenous expression versus those with overexpression

  • Utilize ADRA2A overexpression systems via lentiviral infection, as described in the literature using constructs like EX-Z5688-Lv103 with appropriate empty vector controls (EX-NEG-Lv103)

• Epitope competition:

  • Pre-incubate antibody with the immunizing peptide (if available)

  • For some ADRA2A antibodies, the immunogen is a recombinant fusion protein containing amino acids 240-380 of human ADRA2A (NP_000672.3)

  • Compare signal with and without peptide competition

• Cross-reactivity assessment:

  • Test reactivity against related receptors (other adrenergic receptors)

  • Validate across multiple species if claiming multi-species reactivity

  • Document observed molecular weight (approximately 51 kDa) versus calculated molecular weight (49 kDa)

• Methodological validation:

  • Employ multiple antibodies targeting different epitopes of ADRA2A

  • Compare results across different detection methods (WB, IHC, IF)

  • Ensure appropriate controls for each application (positive and negative)

• Reproducibility verification:

  • Replicate findings across independent experiments

  • Validate batch-to-batch consistency if using different lots

This rigorous validation approach ensures that experimental findings can be confidently attributed to ADRA2A-specific detection rather than non-specific antibody binding.

How can ADRA2A antibodies be utilized in cancer research, particularly for pancreatic cancer studies?

ADRA2A antibodies have shown significant utility in pancreatic cancer research, where they've helped establish ADRA2A's role as a suppressor of the aggressive basal-like/squamous subtype of pancreatic ductal adenocarcinoma (PDAC) . For pancreatic cancer studies, the following methodological approaches are recommended:

• Expression analysis in patient cohorts:

  • Immunohistochemistry on tissue microarrays using ADRA2A antibodies (recommended dilution 1:200)

  • Quantify expression using standardized scoring systems combining intensity and prevalence metrics

  • Correlate expression with clinical parameters (lymph node metastasis, pathological grade, disease stage, and survival)

• Functional studies in cell models:

  • Generate stable ADRA2A-overexpressing PDAC cell lines using lentiviral expression systems

  • The literature documents successful use of ADRA2A constructs (EX-Z5688-Lv103) with appropriate empty vector controls (EX-NEG-Lv103)

  • Select stable clones using puromycin (4 μg/ml)

  • Assess phenotypic changes (invasion capacity, proliferation, gene expression signatures)

• Molecular subtyping:

  • Combine ADRA2A expression analysis with transcriptome profiling to distinguish between classical/progenitor and basal-like/squamous subtypes

  • Correlate ADRA2A levels with molecular signature expressions

• Metabolomic integration:

  • Link ADRA2A expression levels to metabolic profiles, particularly amino acid and carnitine/acylcarnitine metabolism

  • Investigate how ADRA2A modulation affects the metabolic characteristics of different PDAC subtypes

These methodological approaches can provide comprehensive insights into ADRA2A's role in pancreatic cancer progression and its potential as a diagnostic or therapeutic target.

What methods are available for studying ADRA2A interactions with other receptors or signaling molecules?

Investigating ADRA2A interactions with other receptors or signaling molecules requires sophisticated methodological approaches. Based on the literature, the following methods have been successfully employed:

• Co-immunoprecipitation (Co-IP):

  • Express tagged versions of ADRA2A (e.g., HA-tagged) and potential interaction partners (e.g., Flag-tagged)

  • Precipitate one protein and probe for the co-precipitation of the other

  • Include appropriate controls to validate specific interactions

• ELISA-based interaction assays:

  • Co-express differentially tagged proteins (N-terminally HA-tagged ADRA2A and C-terminally Flag-tagged partners, or vice versa)

  • Use robust cell systems like COS-7 cells that can withstand multiple washing steps

  • Include validated positive controls (e.g., growth hormone secretagogue receptor homo-oligomers)

• Proximity-based approaches:

  • Proximity Ligation Assay (PLA) for detecting protein interactions in situ

  • Förster Resonance Energy Transfer (FRET) or Bioluminescence Resonance Energy Transfer (BRET) for live-cell interaction studies

  • Bimolecular Fluorescence Complementation (BiFC) for visualizing protein complexes

• Functional interaction studies:

  • Signal transduction assays (cAMP measurements, given ADRA2A's role in suppressing adenylyl cyclase)

  • Calcium imaging to assess functional coupling

  • Receptor trafficking and internalization studies using fluorescently tagged constructs

• In silico approaches:

  • Molecular docking to predict interaction interfaces

  • Molecular dynamics simulations to assess stability of predicted complexes

  • Integration with structural data where available

These methodological approaches provide complementary information about ADRA2A interactions, enabling a comprehensive understanding of its signaling networks and potential therapeutic targeting strategies.

How can researchers effectively use ADRA2A antibodies in neuroscience research?

ADRA2A plays critical roles in the central nervous system, particularly in inhibiting sympathetic nerve flow and mediating sedation, anxiolysis, and antinociception . For neuroscience applications, the following methodological approaches with ADRA2A antibodies are recommended:

• Neuroanatomical studies:

  • Immunohistochemistry on brain sections using ADRA2A antibodies (1:200-1:800 dilution)

  • Mouse brain tissue has been validated as a positive control

  • Perform antigen retrieval with TE buffer pH 9.0 or alternatively with citrate buffer pH 6.0

  • Use double-labeling with neuronal, glial, or synaptic markers to determine cellular and subcellular localization

• Synaptic physiology:

  • Immunofluorescence to visualize ADRA2A distribution at synapses

  • Co-localization studies with pre- and post-synaptic markers

  • Correlation with electrophysiological recordings to link receptor expression to functional outcomes

• Signaling pathway analysis:

  • Western blotting to assess ADRA2A expression levels and post-translational modifications

  • Phospho-specific antibodies to monitor downstream signaling events

  • Assessment of G-protein coupling and adenylyl cyclase inhibition

• Developmental studies:

  • Track ADRA2A expression patterns during neurodevelopment

  • Correlate with the emergence of specific neuronal circuits and behaviors

  • Combine with transgenic models to understand developmental functions

• Neurological and psychiatric disorder models:

  • Compare ADRA2A expression and localization in disease models versus controls

  • Correlate with behavioral phenotypes

  • Assess effects of pharmacological targeting (agonists/antagonists) on receptor expression and localization

These approaches enable detailed characterization of ADRA2A's distribution, function, and regulation in the nervous system, contributing to our understanding of its role in normal physiology and neurological disorders.