ADCY3 Antibody

What is ADCY3 and why is it significant in research?

ADCY3 (Adenylate Cyclase 3) is a membrane-associated protein that catalyzes the conversion of ATP to cyclic AMP (cAMP), functioning as a critical second messenger in various signaling pathways. ADCY3 has gained significant research interest due to its roles in olfactory signal transduction, metabolism regulation, and potential implications in obesity and cancer development. Studies have demonstrated that ADCY3 is widely expressed in human tissues and plays pivotal roles in modulating cellular responses through cAMP-dependent signaling pathways .

What criteria should researchers consider when selecting an ADCY3 antibody?

When selecting an ADCY3 antibody, researchers should consider:

• Target specificity: Verify the antibody recognizes the specific epitope/region of ADCY3 relevant to your research question. Different antibodies target various domains (e.g., C-terminus, transmembrane domains, or specific amino acid sequences) .

• Host species and isotype: Rabbit polyclonal antibodies are common for ADCY3 detection, but consider experimental compatibility with other reagents .

• Validated applications: Ensure the antibody has been validated for your specific application (WB, IHC, IF, IP, etc.) .

• Cross-reactivity profile: Check reactivity across species (human, mouse, rat, canine) if working with animal models .

• Molecular weight detection: ADCY3 typically appears at 129-130 kDa (calculated) but may show additional bands at different weights (e.g., 82 kDa) depending on tissue type and post-translational modifications .

How do ADCY3 antibodies differ in their epitope recognition and applications?

ADCY3 antibodies vary significantly in their target epitopes and subsequent applications:

Different epitope recognition can provide complementary information about protein structure, localization, and interactions in experimental systems .

What are the optimal conditions for Western blot detection of ADCY3?

For optimal Western blot detection of ADCY3:

• Sample preparation: Use RIPA buffer supplemented with protease inhibitors for protein extraction .

• Protein loading: Load 20-30 μg of protein per lane for optimal detection .

• Gel selection: Use 4-12% gradient gels (such as NuPAGE Bis-Tris) for better resolution of this large protein (129-130 kDa) .

• Transfer conditions: Employ semi-dry transfer systems with PVDF membranes for efficient transfer of this high molecular weight protein .

• Antibody dilutions: Optimal dilutions vary by antibody source but generally range from 1:5000-1:50000 for Western blot applications .

• Detection system: Enhanced chemiluminescence (ECL) substrates provide sufficient sensitivity .

• Controls: Include GAPDH or another appropriate loading control, and positive control tissue (mouse kidney tissue shows strong ADCY3 expression) .

The presence of both 130 kDa and 82 kDa bands may be observed depending on tissue type and experimental conditions .

How should immunofluorescence experiments with ADCY3 antibodies be optimized?

For optimal immunofluorescence results with ADCY3 antibodies:

• Fixation method: Paraformaldehyde (4%) fixation is generally recommended for membrane proteins like ADCY3.

• Permeabilization: Use 0.1-0.3% Triton X-100 for adequate membrane permeabilization to access intracellular epitopes.

• Blocking: 5% normal serum (matching secondary antibody species) with 1% BSA reduces background.

• Antibody dilution: For immunofluorescence/immunocytochemistry, use dilutions ranging from 1:200-1:800 for most ADCY3 antibodies , or 1:500-1:1000 for specialized IF applications .

• Validated cell types: MDCK cells and hTERT-RPE1 cells have been validated for ADCY3 detection by IF/ICC .

• Tissue sections: Rat brain tissue has been validated for IF-P (paraffin sections) with antigen retrieval .

• Antigen retrieval: For tissue sections, use TE buffer pH 9.0 or citrate buffer pH 6.0 for optimal epitope exposure .

• Controls: Include negative controls (secondary antibody only) and positive controls (tissues with known expression).

How can researchers troubleshoot non-specific binding with ADCY3 antibodies?

To troubleshoot non-specific binding issues with ADCY3 antibodies:

• Antibody titration: Perform dilution series to identify optimal antibody concentration that maximizes specific signal while minimizing background (recommended ranges: 1:5000-1:50000 for WB, 1:20-1:200 for IHC, 1:200-1:800 for IF/ICC) .

• Blocking optimization: Increase blocking agent concentration (5-10% normal serum or BSA) and duration (1-2 hours at room temperature).

• Secondary antibody controls: Include controls without primary antibody to identify non-specific secondary antibody binding.

• Washing stringency: Increase washing steps (5-6 times) and duration (10 minutes each) with TBS-T or PBS-T.

• Epitope competition: If available, pre-incubate antibody with immunizing peptide to confirm specificity.

• Cross-reactivity assessment: Verify results in knockout/knockdown models or with alternative antibodies targeting different epitopes of ADCY3 .

• Tissue/cell-specific optimization: Adjust protocols for specific sample types, as ADCY3 detection may require different conditions in different tissues .

How do ADCY3 protein levels correlate with functional activity in experimental systems?

ADCY3 protein levels may not always directly correlate with functional activity for several reasons:

• Post-translational modifications: ADCY3 function is regulated by phosphorylation and other modifications that affect activity without changing protein levels.

• Functional readouts: cAMP levels provide a direct measure of ADCY3 activity. Studies show that even when ADCY3 protein levels are unchanged, as in Adcy3 mut/mut rats, functional deficits can still be observed through decreased cAMP production .

• Downstream signaling: CRE-luciferase and SRE-luciferase assays can effectively measure ADCY3-mediated signaling. Wild-type ADCY3 expression leads to approximately 2.5-fold increase in CRE-luciferase activity and 3-fold increase in SRE-luciferase activity following forskolin stimulation, while mutant ADCY3 shows significantly reduced activity despite similar protein levels .

• Tissue-specific effects: ADCY3 haploinsufficiency (+/-) shows approximately 50% reduction in both mRNA and protein expression, correlating with metabolic phenotypes in adipose tissue, but the severity of functional impairment may vary across tissues .

• Protein-coding mutations: Mutations in transmembrane domains of ADCY3 can alter function without affecting expression, highlighting the importance of functional assays alongside expression analysis .

How can researchers distinguish between specific ADCY3 isoforms or variants in their experimental systems?

Distinguishing between ADCY3 isoforms or variants requires multiple complementary approaches:

• Molecular weight analysis: ADCY3 typically appears at 129-130 kDa (calculated), but variants may show altered molecular weights. For example, some tissues show an additional band at 82 kDa that may represent a specific isoform or proteolytic fragment .

• Epitope-specific antibodies: Use antibodies targeting different domains of ADCY3 (N-terminal, C-terminal, transmembrane regions) to identify structural differences between variants .

• mRNA analysis: Design primers specific to unique regions of different isoforms for RT-PCR or qPCR quantification, as demonstrated in studies measuring ADCY3 expression in tissues .

• Functional assays: cAMP production assays, CRE-luciferase, and SRE-luciferase reporter systems can reveal functional differences between variants that may not be apparent from expression studies alone .

• Genetic models: Compare wild-type, heterozygous (Adcy3 +/-), and specific mutant models (e.g., Adcy3 mut/mut) to understand the functional impact of genetic alterations .

• Domain-specific analysis: For known mutations in specific domains (e.g., transmembrane mutations), design experiments to specifically assess the function of that domain .

What cellular localization patterns are expected for ADCY3 and how do antibody choices affect visualization?

ADCY3 cellular localization patterns vary by cell type and can be influenced by antibody selection:

• Primary localization: ADCY3 is primarily a membrane-associated protein found in plasma membranes and specialized membrane compartments .

• Tissue-specific patterns:

  • In olfactory neurons: Concentrated in cilia

  • In kidney cells: Plasma membrane and potentially cytoplasmic vesicles

  • In neurons: Dendritic processes and potentially postsynaptic densities

• Antibody considerations for localization studies:

  • Antibodies targeting extracellular domains (e.g., 3rd extracellular loop, AA 285-299) may be better for non-permeabilized surface staining

  • Antibodies against intracellular domains require effective permeabilization for access

  • C-terminal antibodies may reveal different localization patterns than N-terminal antibodies if processing or interactions mask epitopes

• Validated detection:

  • hTERT-RPE1 cells show reproducible ADCY3 localization patterns by IF/ICC

  • MDCK cells have been validated for IF/ICC detection of ADCY3

  • Rat brain tissue sections provide reliable IF-P signals for neuronal localization

• Subcellular markers: Co-staining with organelle markers (plasma membrane, Golgi, endosomes) can help precisely define ADCY3 localization in different cell types.

How do mutations in ADCY3 affect protein function and what methods best capture these changes?

Mutations in ADCY3 can affect protein function through various mechanisms that require specific methods for assessment:

• Transmembrane domain mutations: Mutations in the 2nd transmembrane helix, as identified in both rats and humans, can significantly impair protein function without altering expression levels. These require functional rather than expression-based assays .

• Functional assessment methods:

  • cAMP measurement: Direct quantification of cAMP levels using ELISA assays can detect approximately 3-fold increases in wild-type ADCY3 activation versus significantly reduced responses in mutant variants

  • CRE-luciferase reporter assays: Measures cAMP-responsive element activation, showing 2.5-fold increases with wild-type but minimal response with mutant ADCY3

  • SRE-luciferase reporter assays: Detects MAPK/ERK pathway activation, revealing 3-fold increases with wild-type versus diminished signaling with mutant ADCY3

• Protein-protein interactions: Co-immunoprecipitation studies using ADCY3 antibodies (0.5-4.0 μg for 1.0-3.0 mg lysate) can identify altered protein interactions resulting from mutations .

• Signal transduction: Forskolin stimulation experiments reveal that wild-type ADCY3 significantly enhances both cAMP production and downstream transcriptional activation, while mutant ADCY3 shows markedly impaired functionality in these processes .

• Phenotypic consequences: In vivo studies demonstrate that even heterozygous Adcy3 (+/-) and transmembrane mutants (Adcy3 mut/mut) both lead to increased adiposity, though through different mechanisms—increased food intake in males versus decreased energy expenditure in females .

What is the relationship between ADCY3 expression and pathophysiological conditions, and how can antibodies help elucidate these connections?

ADCY3 expression has been linked to several pathophysiological conditions, with antibodies providing crucial insights:

• Obesity and metabolic disorders:

  • Adcy3 heterozygous null mice display increased visceral adiposity without hyperphagia

  • High-fat diet decreases Adcy3 expression in white adipose tissue, liver, and muscle

  • Antibody-based Western blot analysis reveals approximately 50% reduced ADCY3 expression in metabolic tissues of heterozygous models

  • Sex-specific differences in obesity mechanisms (food intake vs. energy expenditure) can be tracked using tissue-specific ADCY3 immunostaining

• Cancer development:

  • ADCY3 overexpression increases cell migration (by 43%), invasion (4.95-fold), viability, and clonogenicity in cancer models

  • Silencing ADCY3 in gastric cancer cells reduces these tumorigenic properties

  • Antibody techniques (Western blot, IHC) help correlate ADCY3 expression levels with Lauren's intestinal-type gastric cancers

• Neuropsychiatric conditions:

  • Adcy3 mut/mut males display increased passive coping and decreased memory while females show increased anxiety-like behavior

  • Immunofluorescence analysis of brain tissue can map ADCY3 distribution in neural circuits related to these behaviors

• Methodological applications:

  • Immunohistochemistry (1:20-1:200 dilution) can localize ADCY3 expression in disease-relevant tissues

  • Western blot (1:5000-1:50000 dilution) quantifies expression changes across disease models

  • Immunoprecipitation isolates ADCY3 protein complexes that may be altered in disease states

How do sex differences influence ADCY3 function and expression, and what experimental considerations are important when studying these differences?

Research has revealed important sex-specific differences in ADCY3 function and expression that require careful experimental design:

• Metabolic phenotypes:

  • In Adcy3 mut/mut and Adcy3 +/- rats, both sexes develop increased adiposity, but through different mechanisms

  • Males: Increased food intake drives weight gain

  • Females: Decreased energy expenditure is the primary driver

• Behavioral impacts:

  • Males with Adcy3 mutations display increased passive coping and decreased memory

  • Females show predominantly increased anxiety-like behavior

  • These distinctions highlight the importance of analyzing both sexes separately in behavioral experiments

• Experimental considerations:

  • Sample size calculation: Power analyses should account for potentially higher variability in female subjects due to estrous cycle effects

  • Hormonal influences: Consider estrous cycle staging in females or hormone measurements

  • Statistical analysis: Always analyze data with sex as a factor first before stratifying by sex, as demonstrated in studies showing multiple significant sex effects and genotype-by-sex interactions

  • Metabolic measurements: Adjust parameters like flow rates for metabolic cages differently for males and females due to body size differences

• Physiological mechanisms:

  • Energy expenditure shows significantly decreased average daily and cumulative energy expenditure in Adcy3 mut/mut females (p=0.0264 and p=0.0324) and Adcy3 +/- females (p=0.0068 and p=0.0265), but no differences in males

  • Respiratory exchange ratio (RER) shows increases in Adcy3 +/- males (p=0.0100) but not females

• Technical approaches:

  • Use sex-balanced experimental designs

  • Include sufficient animals of each sex (17-20 rats/group recommended for metabolic experiments)

  • Report sex-specific findings separately rather than pooling data

How can researchers effectively combine ADCY3 antibody techniques with genetic manipulation models to establish causality?

Researchers can establish causality by integrating antibody techniques with genetic models through several strategic approaches:

• Complementary model systems:

  • Complete knockout models (though homozygous ADCY3 KO may be lethal shortly after birth)

  • Heterozygous models (Adcy3 +/-) showing approximately 50% protein expression reduction

  • Protein-coding mutation models (Adcy3 mut/mut) with normal expression but altered function

  • Tissue-specific conditional knockouts to isolate effects in specific cell populations

• Validation strategy:

  • Confirm genetic manipulation success using antibody-based techniques (Western blot, IHC)

  • Analyze both mRNA (qPCR) and protein (Western blot) levels to ensure concordance

  • Compare protein expression patterns across genotypes using standardized immunofluorescence protocols

• Rescue experiments:

  • Re-express wild-type ADCY3 in knockout backgrounds and confirm restoration using antibodies

  • Compare phenotypes of different genetic models (complete KO vs. mutations) using standardized antibody-based readouts

• Functional correlation:

  • Link ADCY3 expression levels (detected by antibodies) with functional readouts (cAMP levels, reporter assays)

  • Correlate tissue-specific expression patterns with phenotypic manifestations using antibody-based tissue analysis

• Technical considerations:

  • Use consistent antibody dilutions across experiments (e.g., 1:5000-1:50000 for WB, 1:20-1:200 for IHC)

  • Include appropriate positive and negative controls from validated tissues (mouse kidney, human heart tissue)

  • Consider both expression levels and subcellular localization in interpreting results

What are the technical challenges in measuring ADCY3 activity versus expression, and how can researchers address these challenges?

Measuring ADCY3 activity presents distinct challenges compared to measuring expression:

• Expression vs. activity discrepancies:

  • Protein-coding mutations may not affect expression levels but significantly impair function

  • Post-translational modifications can alter activity without changing expression levels

  • Protein interactions may inhibit or enhance activity independently of expression

• Direct activity measurement approaches:

  • cAMP assays: Quantify intracellular cAMP levels as a direct measure of adenylyl cyclase activity

  • Challenge with activators: Forskolin stimulation reveals functional capacity of ADCY3

  • Reporter systems: CRE-luciferase and SRE-luciferase assays measure downstream pathway activation

• Technical solutions:

  • Paired measurements: Always assess both protein levels (Western blot, 1:5000-1:50000 dilution) and functional readouts in the same experimental system

  • Time-course experiments: Examine both acute and sustained ADCY3 activation

  • Subcellular fractionation: Isolate membrane fractions where ADCY3 is functionally active

  • Pathway inhibitors: Use specific inhibitors to confirm observed effects are ADCY3-dependent

• Quality control measures:

  • Positive controls: Include forskolin as a direct adenylyl cyclase activator

  • Negative controls: Use inhibitors of cAMP signaling or ADCY3-deficient models

  • Parallel measurements: Assess multiple parameters (cAMP, PKA activity, CREB phosphorylation)

• Comparative analysis:

  • Wild-type ADCY3 typically shows approximately 3-fold increases in cAMP levels after stimulation

  • CRE-luciferase activity increases approximately 2.5-fold with wild-type ADCY3

  • SRE-luciferase shows nearly 3-fold enhancement with functional ADCY3

  • Significant reductions in these metrics indicate functional impairment even when expression appears normal