ADRA2C Antibody

What is ADRA2C and why is it relevant to neuroscience research?

ADRA2C (alpha-2C adrenergic receptor) is a member of the G protein-coupled receptor superfamily that mediates catecholamine-induced inhibition of adenylate cyclase through G proteins . It plays critical roles in regulating neurotransmitter release from sympathetic nerves and adrenergic neurons. ADRA2C is particularly relevant to neuroscience research because of its involvement in various neuropsychiatric conditions, including schizophrenia, where significant alterations in expression have been documented . When designing experiments targeting ADRA2C, researchers should consider its distribution in brain regions, particularly the dorsolateral prefrontal cortex (DLPFC), where its expression has been extensively studied in relation to schizophrenia and antipsychotic treatment effects .

What are the common applications for ADRA2C antibodies in research?

ADRA2C antibodies are validated for multiple experimental applications with appropriate methodological considerations for each:

When selecting applications, researchers should consider which approach will best answer their specific research question. Western blotting provides quantitative protein expression data, while immunohistochemistry offers spatial information about receptor distribution in tissue contexts .

How should samples be prepared for optimal ADRA2C antibody detection?

For formalin-fixed paraffin-embedded (FFPE) tissue samples used in IHC-P applications, heat-induced antigen retrieval is essential for unmasking ADRA2C epitopes . This typically involves treating tissue sections with a citrate or EDTA buffer at elevated temperatures. For Western blot applications, standard protein extraction protocols using RIPA or similar buffers are appropriate, with particular attention to membrane protein enrichment techniques since ADRA2C is a transmembrane receptor .

To maximize detection sensitivity while maintaining specificity, researchers should optimize protein loading (typically 20-50 μg total protein for Western blots) and antibody dilutions. Storage of antibody solutions should follow manufacturer recommendations: for short-term use (up to one month), storage at 4°C is acceptable, while long-term storage requires -20°C with avoidance of repeated freeze-thaw cycles .

How can I optimize Western blot protocols for ADRA2C detection?

When optimizing Western blot protocols for ADRA2C detection, researchers should first consider the observed molecular weight discrepancy. While the calculated molecular weight of ADRA2C is 49.5 kDa, it is typically observed at 39 kDa on Western blots . This difference may result from post-translational modifications or protein processing.

For optimal results:

• Sample preparation: Use membrane protein enrichment methods since ADRA2C is a transmembrane receptor

• Protein denaturation: Heat samples at 95°C for 5 minutes in Laemmli buffer containing SDS and a reducing agent

• Gel percentage: Use 10-12% polyacrylamide gels for optimal resolution in the 39-50 kDa range

• Transfer conditions: Optimize transfer time and voltage for membrane proteins

• Blocking: Use 5% non-fat dry milk or BSA in TBST

• Antibody dilution: Start with 1:1000 dilution and adjust based on signal strength

• Controls: Include positive controls (brain tissue lysates) and negative controls (tissues known not to express ADRA2C)

For quantitative analysis, normalization to housekeeping proteins like GAPDH or beta-actin is essential, similar to the approach used in mRNA expression studies of ADRA2C where reference genes are employed for accurate quantification .

What considerations are important for immunohistochemical detection of ADRA2C?

For successful immunohistochemical detection of ADRA2C in tissue sections:

• Fixation protocol: Standard 10% neutral buffered formalin fixation is compatible with available ADRA2C antibodies

• Antigen retrieval: Heat-induced antigen retrieval is critical - typically using citrate buffer (pH 6.0) or EDTA buffer (pH 9.0)

• Background reduction: Include a peroxidase blocking step and appropriate serum blocking

• Antibody dilution: Begin with manufacturer-recommended dilutions and optimize as needed

• Incubation conditions: Overnight incubation at 4°C often yields better results than shorter room temperature incubations

• Detection systems: HRP-polymer detection systems generally provide good sensitivity with low background

• Counterstaining: Hematoxylin provides good nuclear contrast without obscuring specific staining

When interpreting IHC results, researchers should be aware that ADRA2C shows predominant expression in specific regions, such as smooth muscle cells in human arteries, as demonstrated in validated IHC applications .

How do I select appropriate controls for ADRA2C antibody validation?

Proper controls are essential for validating ADRA2C antibody specificity:

Positive controls:

• Human artery smooth muscle tissue (validated for IHC-P)

• Brain tissue from human, mouse, or rat (particularly cortical regions)

• Cell lines with known ADRA2C expression

Negative controls:

• Primary antibody omission

• Isotype controls (rabbit IgG at matching concentration)

• Tissues with confirmed absence of ADRA2C expression

• Antibody pre-absorption with immunizing peptide (when available)

Technical validation approaches:

• Comparison of multiple antibodies targeting different epitopes of ADRA2C

• Correlation of protein detection with mRNA expression data

• Knockdown or knockout validation in appropriate cell lines

• Cross-validation with different detection techniques (e.g., IF, WB, IHC)

This comprehensive validation strategy ensures that experimental observations truly reflect ADRA2C biology rather than non-specific binding or artifacts .

How can ADRA2C antibodies be used to study epigenetic regulation of receptor expression?

ADRA2C expression is subject to epigenetic regulation, particularly through histone modifications at the promoter region. Research has identified both permissive (H3K4me3, H3ac, H3K9ac, H3K27ac, H4K5ac, H4K16ac) and repressive (H3K27me3) histone post-translational modifications (PTMs) at the ADRA2C promoter .

To study these mechanisms:

• Combine ADRA2C antibody-based protein detection with chromatin immunoprecipitation (ChIP) assays targeting specific histone modifications at the ADRA2C promoter

• Design experimental protocols that integrate:

  • Western blot quantification of ADRA2C protein levels

  • RT-qPCR measurement of ADRA2C mRNA expression

  • ChIP analysis of histone modifications at the ADRA2C promoter

  • Treatment with epigenetic modifiers (HDAC inhibitors, HMT inhibitors)

Recent research has demonstrated that the ADRA2C promoter region shows differential histone modification patterns in schizophrenia, with upregulation of ADRA2C expression (+53%) observed regardless of antipsychotic treatment . This suggests that targeting the epigenetic regulators of ADRA2C may represent a novel therapeutic approach worth investigating.

What approaches can be used to study ADRA2C in the context of antipsychotic drug effects?

Studies have revealed differential effects of antipsychotic drugs on ADRA2A and ADRA2C expression, providing important insights for neuropsychiatric research. While ADRA2A mRNA expression was selectively upregulated in antipsychotic-treated schizophrenia subjects (+93%), ADRA2C showed increased expression in schizophrenia regardless of treatment status .

To investigate ADRA2C in relation to antipsychotic effects:

• Design experiments comparing antipsychotic-treated and untreated conditions:

  • In vitro: Neuronal cell cultures treated with various antipsychotics

  • In vivo: Animal models receiving acute vs. chronic antipsychotic administration

  • Human studies: Post-mortem brain tissue from treated vs. untreated patients

• Implement a multi-level analysis approach:

  • Protein expression: Western blot and IHC with ADRA2C antibodies

  • mRNA quantification: RT-qPCR normalized to reference genes like GAPDH and RPS13

  • Receptor functionality: Second messenger assays measuring adenylate cyclase inhibition

  • Signaling pathway analysis: Downstream G-protein activation

• Use the ΔΔCt method for mRNA quantification as demonstrated in research:
  ΔΔCt = (Ct(target gene)sample – Ct(reference gene)sample) – (Ct(target gene)reference – Ct(reference gene)reference)
  Relative expression = 2^-ΔΔCt

This methodological approach has revealed that clozapine treatment in rats increases Adra2c mRNA expression without affecting Adra2a, suggesting differential regulation of these receptor subtypes by antipsychotic drugs .

How can co-localization studies be designed to examine ADRA2C interactions with other proteins?

Co-localization studies exploring ADRA2C interactions require careful experimental design:

• Double immunofluorescence labeling:

  • Use ADRA2C antibodies in combination with antibodies against potential interaction partners

  • Select antibodies raised in different host species (e.g., rabbit anti-ADRA2C with mouse anti-target protein)

  • Employ spectrally distinct fluorophore-conjugated secondary antibodies

  • Validate specificity with appropriate controls

• Proximity ligation assays (PLA):

  • More sensitive than traditional co-localization for detecting protein-protein interactions

  • Generates fluorescent signal only when proteins are within 40nm

  • Requires optimization of antibody dilutions and PLA probe concentrations

• Co-immunoprecipitation (Co-IP):

  • Complements imaging approaches with biochemical evidence

  • Requires careful selection of lysis conditions to preserve membrane protein interactions

  • Western blot detection using validated ADRA2C antibodies

  • Controls should include reverse Co-IP and IgG controls

• FRET/BRET approaches:

  • For living cell studies of dynamic interactions

  • Requires generation of fluorescent/luminescent protein fusion constructs

These approaches can help elucidate ADRA2C's interactions with G proteins, regulators of G protein signaling (RGS), and other components of adrenergic signaling pathways, providing mechanistic insights into how ADRA2C mediates inhibition of adenylate cyclase .

How should I address inconsistencies between observed and predicted molecular weight for ADRA2C?

The discrepancy between the calculated molecular weight of ADRA2C (49.5 kDa) and its observed migration pattern on SDS-PAGE (39 kDa) is a common challenge in ADRA2C research . This difference may result from several factors:

• Post-translational modifications (PTMs):

  • Glycosylation status can significantly affect migration patterns

  • Phosphorylation, ubiquitination, or other PTMs may alter mobility

  • Enzymatic deglycosylation experiments can determine glycosylation contribution

• Protein processing:

  • Proteolytic cleavage of the full-length protein

  • Alternative splicing resulting in shorter protein isoforms

  • N-terminal or C-terminal truncations

• Technical considerations:

  • Sample preparation conditions (reducing vs. non-reducing)

  • Gel percentage and running conditions

  • Protein standards used for molecular weight estimation

When troubleshooting:

• Compare migration patterns across different tissue/cell types

• Use multiple antibodies targeting different epitopes

• Employ mass spectrometry to confirm protein identity

• Consider analyzing mRNA expression of potential splice variants

Researchers should report both the observed and predicted molecular weights in publications, along with hypotheses explaining the discrepancy .

What are common pitfalls in ADRA2C antibody-based experiments and how can they be avoided?

Common pitfalls in ADRA2C antibody experiments include:

• Non-specific binding:

  • Use proper blocking (5% BSA or milk)

  • Optimize antibody dilutions (starting with 1:500-1:2000 for WB)

  • Include appropriate negative controls

  • Consider pre-absorption with immunizing peptide when available

• Poor signal-to-noise ratio:

  • For IHC: Implement proper antigen retrieval (heat-induced)

  • For WB: Optimize protein loading (20-50 μg)

  • Use fresh antibody aliquots to avoid degradation

  • Consider signal amplification methods for low-abundance targets

• Inconsistent results between experiments:

  • Standardize protocols with detailed SOPs

  • Use the same lot of antibody when possible

  • Implement positive controls in every experiment

  • Maintain consistent sample processing

• Cross-reactivity with other alpha-2 adrenergic receptors:

  • ADRA2A, ADRA2B, and ADRA2C share homology

  • Verify antibody specificity against all three subtypes

  • Consider epitope mapping to identify uniquely targeted regions

• Storage and handling:

  • Follow manufacturer guidelines for temperature (4°C short-term, -20°C long-term)

  • Avoid repeated freeze-thaw cycles

  • Properly mix antibody solution before use (gentle inversion, not vortexing)

  • Check for visible precipitation before use

By systematically addressing these potential issues, researchers can significantly improve the reliability and reproducibility of their ADRA2C antibody-based experiments.

How can I differentiate between ADRA2C and other alpha-2 adrenergic receptor subtypes in my experiments?

Differentiating between the highly homologous alpha-2 adrenergic receptor subtypes (ADRA2A, ADRA2B, and ADRA2C) requires careful methodology:

• Antibody selection:

  • Choose antibodies raised against unique epitopes of ADRA2C

  • Review epitope mapping data or sequence alignment information

  • Verify specificity against all three subtypes when possible

  • Consider monoclonal antibodies for higher specificity

• Experimental validation:

  • Test antibodies on tissues with known differential expression patterns

  • Use knockout/knockdown models when available

  • Compare with subtype-specific pharmacological tools

  • Implement siRNA knockdown of individual subtypes to confirm specificity

• Complementary approaches:

  • Combine protein detection with mRNA analysis

  • Use RT-qPCR with subtype-specific primers

  • Consider subtype-selective ligands in functional assays

  • Implement receptor binding studies with selective compounds

• Data interpretation:

  • Be aware of species differences in expression patterns

  • Consider developmental and disease-state changes in expression

  • Document the specific region/tissue examined (e.g., DLPFC in schizophrenia studies)

  • Report antibody validation methods in publications

These methodological considerations help ensure that observations attributed to ADRA2C are not confounded by cross-reactivity with other alpha-2 adrenergic receptor subtypes.

How are ADRA2C antibodies used in schizophrenia research?

ADRA2C antibodies have become valuable tools in schizophrenia research, where significant alterations in adrenergic signaling have been documented. Recent studies using these antibodies have revealed:

• Expression changes:

  • ADRA2C mRNA expression is upregulated (+53%) in schizophrenia regardless of antipsychotic treatment

  • This contrasts with ADRA2A, which shows selective upregulation (+93%) only in antipsychotic-treated subjects

• Methodological approaches:

  • Immunohistochemistry to map receptor distribution in the dorsolateral prefrontal cortex (DLPFC)

  • Western blot quantification to measure protein level changes

  • Combined with RT-qPCR for correlation between protein and mRNA levels

  • Integration with epigenetic studies examining histone modifications at the ADRA2C promoter

• Experimental designs:

  • Comparisons between schizophrenia subjects and matched controls (n=24 pairs)

  • Stratification by antipsychotic treatment status (AP-free n=12 vs. AP-treated n=12)

  • Parallel animal studies using acute and chronic antipsychotic administration

  • Multi-level analysis integrating mRNA, protein, and epigenetic data

These studies suggest that altered ADRA2C expression may represent a primary pathophysiological feature of schizophrenia rather than a medication effect, making it a potential biomarker or therapeutic target worthy of further investigation .

What methodological considerations are important when studying epigenetic regulation of ADRA2C?

Studies of epigenetic regulation of ADRA2C require careful methodological approaches:

• Chromatin immunoprecipitation (ChIP) methodology:

  • Target both permissive (H3K4me3, H3ac, H3K9ac, H3K27ac, H4K5ac, H4K16ac) and repressive (H3K27me3) histone marks

  • Implement rigorous controls including input DNA and IgG controls

  • Design primers specifically targeting the ADRA2C promoter region

  • Consider chromatin accessibility assays (ATAC-seq) as complementary approaches

• Integration with expression data:

  • Correlate histone modifications with ADRA2C mRNA levels

  • Normalize mRNA expression using reference genes (GAPDH, RPS13)

  • Use the ΔΔCt method for relative quantification:
    2^-ΔΔCt where ΔΔCt = (Ct(ADRA2C)sample – Ct(reference)sample) – (Ct(ADRA2C)reference – Ct(reference)reference)

• Bivalent chromatin considerations:

  • Research has identified bivalent chromatin at the ADRA2C promoter in schizophrenia

  • This is characterized by co-occurrence of permissive H3K4me3 and repressive H3K27me3

  • Enhanced H4K16ac at the ADRA2C promoter may trigger upregulation

• Experimental manipulations:

  • HDAC inhibitors to investigate the role of histone acetylation

  • HMT inhibitors to explore histone methylation effects

  • Compare effects between neuronal and non-neuronal cells

  • Assess the impact of antipsychotic drugs on epigenetic patterns

These approaches have revealed that epigenetic mechanisms differentially modulate ADRA2C expression in schizophrenia, potentially explaining the observed upregulation regardless of antipsychotic treatment status .

How can ADRA2C antibodies contribute to developing novel therapeutic approaches?

ADRA2C antibodies play crucial roles in research that may lead to novel therapeutic approaches:

• Target validation studies:

  • Confirm ADRA2C protein expression in relevant tissues

  • Quantify receptor levels in disease states vs. controls

  • Map subcellular localization to inform drug delivery strategies

  • Identify post-translational modifications that might affect drug binding

• Drug discovery applications:

  • Screen for compounds that modulate ADRA2C expression

  • Evaluate effects of potential therapeutics on receptor levels

  • Assess receptor internalization/trafficking in response to drug candidates

  • Monitor receptor expression changes during treatment

• Therapeutic monitoring applications:

  • Develop assays to monitor ADRA2C expression as biomarkers

  • Evaluate treatment effects on receptor expression

  • Correlate receptor levels with clinical outcomes

  • Identify patient subgroups based on receptor expression patterns

• Precision medicine approaches:

  • Stratify patients based on ADRA2C expression profiles

  • Correlate genetic variants with protein expression levels

  • Identify responder/non-responder populations for adrenergic-targeting drugs

  • Design combination therapies targeting ADRA2C and related pathways

The differential upregulation of ADRA2C in schizophrenia regardless of antipsychotic treatment suggests it may represent a disease-associated marker rather than a medication effect, making it a compelling target for novel therapeutic development . Furthermore, the discovery of epigenetic regulation mechanisms offers potential for epigenetic-modifying drugs as a therapeutic strategy for conditions with altered ADRA2C expression.