ADCY6 Antibody

What is ADCY6 and what is its basic function?

ADCY6 is adenylate cyclase type 6, a 130-131 kDa membrane-bound enzyme that catalyzes the conversion of ATP to cyclic AMP (cAMP). It functions as a critical component in G protein-coupled receptor (GPCR) signaling pathways and intracellular signal transduction . The human ADCY6 protein has a canonical length of 1168 amino acids and is predominantly localized to the cell membrane. This enzyme plays essential roles in regulating diverse physiological processes including metabolism, gene expression, and neuronal signaling, which are vital for maintaining homeostasis and responding to environmental changes . As a member of the adenylyl cyclase class-4/guanylyl cyclase protein family, ADCY6 is intricately regulated by G protein-coupled receptors, where binding of an agonist leads to activation and signal amplification .

What applications are ADCY6 antibodies validated for?

ADCY6 antibodies are validated for multiple experimental applications, with performance varying by specific antibody product. The most common applications include:

When designing experiments, researchers should consider that some antibodies may perform better in certain applications than others. For example, antibody 14616-1-AP from Proteintech is extensively validated for WB and IP with specifically documented reactivity in brain tissues . Always verify the validation data for your specific application and tissue of interest before proceeding with experiments.

What species reactivity has been confirmed for ADCY6 antibodies?

Most commercially available ADCY6 antibodies show reactivity across human, mouse, and rat samples. This cross-reactivity is particularly valuable for comparative studies and translational research. According to product validation data:

• Proteintech antibody 14616-1-AP has been tested and confirmed reactive in human, mouse, and rat samples, with specific validation in brain tissues .

• Abbexa's ADCY6 antibody demonstrates reactivity with human, mouse, and rat samples across multiple applications .

• Several other manufacturers including Boster Bio and Santa Cruz Biotechnology confirm their antibodies react with human, mouse, and rat ADCY6 .

When working with other species or specialized tissue samples, preliminary validation experiments are recommended as reactivity may not be guaranteed. The conservation of ADCY6 across mammalian species allows for consistent performance in many experimental models, but specific epitope recognition should be verified when extending to novel applications.

How can I verify antibody specificity for ADCY6 versus other adenylate cyclase isoforms?

Verifying antibody specificity for ADCY6 versus other adenylate cyclase isoforms requires multiple validation approaches:

• Knockout/Knockdown Controls: The gold standard for specificity validation is testing the antibody in ADCY6 knockout or knockdown samples. Several publications have utilized Adcy6-/- models to confirm antibody specificity . In these studies, immunostaining of Adcy6-/- tissues showed absence of signals that were present in wild-type tissues, confirming specificity.

• Cross-Reactivity Assessment: Some antibodies may cross-react with other adenylate cyclase isoforms, particularly ADCY5 due to structural similarity. For instance, the AC5/6 antibody from Santa Cruz recognizes both isoforms . To distinguish between these isoforms:

  • Compare expression patterns with known tissue distribution (ADCY6 is broadly expressed while ADCY5 shows more restricted expression)

  • Use parallel experiments with isoform-specific antibodies

  • Examine molecular weight differences (subtle but may be distinguishable on well-resolved Western blots)

• Immunogen Analysis: Review the immunogen information provided by manufacturers. Antibodies raised against unique regions of ADCY6 will have greater specificity. For example, some antibodies are generated against the N-terminal amino acids 13-27 of rat ADCY6 , while others target the C-terminal region.

To conclusively differentiate between adenylate cyclase isoforms, combine antibody-based detection with complementary techniques such as RT-PCR for isoform-specific mRNA expression or mass spectrometry-based protein identification.

What are the optimal sample preparation methods for different applications?

Sample preparation methods vary significantly depending on the application and tissue type:

For Western Blotting of ADCY6:

• Use fresh tissue or cells when possible, as ADCY6 degradation can occur in improperly stored samples

• Extract proteins using buffers containing protease inhibitors

• Include membrane-protein extraction components such as mild detergents (0.5-1% NP-40 or Triton X-100)

• Sonication may improve extraction efficiency for this membrane-bound protein

• For brain tissue specifically, specialized protocols may be needed to account for high lipid content

For Immunohistochemistry/Immunofluorescence:

• Fixation with 4% paraformaldehyde is typically effective

• Antigen retrieval steps are often necessary - heat-induced epitope retrieval using citrate buffer (pH 6.0) is commonly employed

• For inner ear hair cells, specialized fixation and permeabilization protocols have been validated in studies examining ADCY6 localization at the stereociliary base

For Immunoprecipitation:

• Use 0.5-4.0 μg of antibody per 1.0-3.0 mg of total protein lysate

• Pre-clearing lysates may reduce non-specific binding

• For brain tissue IP, specific protocols have been validated showing successful pulldown of ADCY6

The choice of buffer systems should be compatible with the target tissue and downstream application, with particular attention to preserving membrane protein integrity when studying ADCY6.

How does ADCY6 expression and localization vary across tissues, and what implications does this have for experimental design?

ADCY6 shows distinct expression patterns and subcellular localization across different tissues, which has significant implications for experimental design:

Brain Tissue:

• ADCY6 is expressed in brain tissues of humans, mice, and rats

• RT-PCR analysis from knockout studies shows significant ADCY6 mRNA expression in wild-type brain tissues

• Experimental implication: Brain samples provide reliable positive controls for antibody validation

Renal Tissue:

• ADCY6 is expressed in collecting duct cells and plays a role in arginine vasopressin (AVP)-stimulated renal water reabsorption

• Knockout studies show a 39.5% reduction in ADCY6 mRNA in renal papilla from collecting duct-specific ADCY6 knockout mice

• Experimental implication: When studying renal ADCY6, consider cell-type specific expression and potential compensatory mechanisms

Inner Ear Hair Cells:

• ADCY6 localizes specifically to the basal portion of stereocilia in cochlear inner hair cells, outer hair cells, and vestibular hair cells

• This localization depends on the integrity of the ankle link complex; disruption causes ADCY6 to extend throughout stereocilia

• Experimental implication: When studying ADCY6 in sensory cells, subcellular localization is critical and may be altered in disease states

Retinal Tissue:

• ADCY6 is mainly expressed in retinal cells other than photoreceptors, as demonstrated by comparable expression in wild-type and rd1 retinas (where photoreceptors are lost)

• AC5 rather than AC6 appears to be the predominant adenylate cyclase in photoreceptors

• Experimental implication: When studying retinal tissue, distinguishing between AC5 and AC6 is crucial

Heart Tissue:

• ADCY6 is predominantly expressed in cardiac tissue

• Experimental implication: Heart samples provide reliable positive controls for antibody validation

This tissue-specific expression profile necessitates careful selection of positive and negative controls when designing experiments targeting ADCY6. Additionally, subcellular localization studies require high-resolution imaging techniques to accurately document ADCY6 distribution patterns.

What are the known disease associations of ADCY6 and how can antibodies be used to study these connections?

ADCY6 has been implicated in several pathological conditions, and antibodies serve as critical tools for investigating these disease associations:

Oral Tongue Squamous Cell Carcinoma (OTSCC):

• ADCY6 is downregulated in OTSCC tissue samples and cell lines

• Lower ADCY6 expression correlates with poorer prognosis, TNM stage, and tumor size in OTSCC patients

• Mechanistically, ADCY6 acts as a tumor suppressor by impairing the Hippo signaling pathway

• Research application: Antibodies can be used for prognostic tissue staining, mechanism studies, and therapeutic target validation

Lethal Arthrogryposis Multiplex Congenita:

• Four ADCY6 mutations have been linked to this lethal condition in humans

• These include homozygous missense mutations (Y992C and R1116C) and compound heterozygous missense and splice site mutations

• Research application: Antibodies can help assess how these mutations affect ADCY6 protein expression, localization, and interaction with binding partners

Renal Water Homeostasis Disorders:

• Collecting duct-specific knockout of ADCY6 causes a urine-concentrating defect during water deprivation

• Research application: Antibodies can be used to study ADCY6's role in arginine vasopressin signaling and water channel trafficking

Inner Ear and Retinal Disorders:

• While ADCY6 knockout mice showed normal cochlear and retinal structure and function, ADCY6 localization depends on proteins associated with Usher syndrome

• Research application: Antibodies can help investigate potential indirect roles of ADCY6 in sensory cell pathologies

To effectively study these disease associations, researchers should employ:

• Tissue microarrays with appropriate antibody dilutions to assess expression in patient samples

• Co-localization studies with pathway components to understand mechanistic relationships

• Protein-protein interaction studies combined with antibody-based detection methods

• Functional rescue experiments in disease models with antibody validation of protein restoration

The expression level of ADCY6 appears to have prognostic significance in certain cancers, making quantitative analysis methods particularly valuable in translational research applications.

What experimental controls are essential when using ADCY6 antibodies?

Rigorous experimental controls are essential for generating reliable data with ADCY6 antibodies:

Positive Controls:

• Tissue-specific positive controls: Human and mouse brain tissues have been validated as positive controls for Western blot and immunoprecipitation

• Recombinant protein: Purified human ADCY6 recombinant protein can serve as a positive control, particularly for antibodies targeting amino acids V312-F652

• Overexpression systems: Cells transiently transfected with ADCY6 expression vectors provide strong positive controls

Negative Controls:

• Knockout/knockdown samples: Adcy6-/- tissues or ADCY6 siRNA-treated cells are gold-standard negative controls

• Tissues with minimal expression: Based on expression data, tissues with naturally low ADCY6 expression can serve as partial negative controls

• Primary antibody omission: Essential control for all immunostaining procedures

• Isotype controls: Using matched isotype IgG (rabbit IgG for polyclonal antibodies) at the same concentration

Specificity Controls:

• Peptide competition assays: Pre-incubating the antibody with immunizing peptide should abolish specific signals

• Multiple antibody validation: Using more than one antibody targeting different epitopes of ADCY6

• Parallel detection methods: Confirming protein expression with mRNA detection techniques

Application-Specific Controls:

• For Western blot: Loading controls (β-actin, GAPDH) and molecular weight markers to confirm the expected 130-150 kDa band

• For immunoprecipitation: IgG control IP and input sample controls

• For immunofluorescence: Counterstaining with established markers of relevant subcellular structures

In studies of inner ear hair cells, researchers have used Adcy6-/- tissues as negative controls while comparing signals to established markers like ADGRV1 to validate specificity and localization patterns . Similarly, in retinal studies, comparing wild-type and Rd1 mice (which lack photoreceptors) helped distinguish cell type-specific expression .

What methodological challenges exist when studying the functional relationship between ADCY6 and its signaling partners?

Investigating functional relationships between ADCY6 and its signaling partners presents several methodological challenges:

Challenges in Studying Protein-Protein Interactions:

• Membrane protein complexes: As a membrane-bound protein, ADCY6 requires specialized approaches for studying interactions

• Complex stability: ADCY6 interactions with G proteins and other signaling molecules may be transient or condition-dependent

• Antibody interference: Antibodies may disrupt native protein interactions or fail to recognize complexed ADCY6

Recommended Solutions:

• Use crosslinking approaches prior to immunoprecipitation

• Employ proximity ligation assays to detect in situ interactions

• Consider membrane-compatible co-IP protocols with mild detergents

Challenges in Distinguishing Between Adenylate Cyclase Isoforms:

• High sequence homology: ADCY5 and ADCY6 share significant sequence similarity, complicating isoform-specific studies

• Co-expression in tissues: Multiple AC isoforms may be expressed in the same tissue

• Functional redundancy: Knockdown of one isoform may be compensated by others

Recommended Solutions:

• Use isoform-specific antibodies when available

• Combine with genetic approaches (siRNA, CRISPR) targeting specific isoforms

• Design experiments based on known differential regulation (e.g., ADCY6 is inhibited by specific Ca²⁺ concentrations)

Challenges in Signaling Pathway Analysis:

• Temporal dynamics: cAMP signaling occurs rapidly and may be difficult to capture

• Spatial organization: ADCY6 signaling may be compartmentalized within cells

• Multi-pathway interactions: ADCY6 interfaces with multiple signaling networks (e.g., Hippo pathway in cancer )

Recommended Solutions:

• Use live-cell cAMP sensors for temporal studies

• Apply super-resolution microscopy to study spatial organization

• Employ pathway inhibitors to dissect specific connections

In inner ear research, investigators faced challenges distinguishing the function of ADCY6 from other ACs and addressed this through comprehensive knockout studies and detailed localization experiments . Similarly, in cancer research, researchers studying ADCY6's role in the Hippo pathway used cell function recovery tests to investigate the mechanism of ADCY6's effect on malignant biological behavior .

What are common troubleshooting strategies for weak or inconsistent ADCY6 antibody signals?

When encountering weak or inconsistent signals with ADCY6 antibodies, several methodological adjustments can improve results:

For Western Blotting:

• Protein extraction optimization:

  • Use specialized membrane protein extraction buffers

  • Include phosphatase inhibitors (ADCY6 may be regulated by phosphorylation)

  • Extend extraction time for membrane-bound proteins

  • Consider gentle sonication to improve solubilization

• Antibody incubation adjustments:

  • Extend primary antibody incubation to overnight at 4°C

  • Optimize antibody concentration through titration (typical range: 1:500-1:2000 )

  • Use 5% BSA instead of milk for blocking and antibody dilution

• Detection enhancement:

  • Use high-sensitivity ECL substrates

  • Consider signal amplification systems for low abundance targets

  • Increase exposure time while monitoring background

For Immunohistochemistry/Immunofluorescence:

• Fixation and antigen retrieval optimization:

  • Test multiple fixatives (PFA, methanol, acetone)

  • Compare different antigen retrieval methods (heat-induced vs. enzymatic)

  • Optimize retrieval buffer pH and incubation time

• Signal amplification:

  • Use biotin-streptavidin systems or tyramide signal amplification

  • Consider polymeric detection systems for IHC

  • Optimize antibody concentration through titration (typical range: 1:100-1:300 )

• Background reduction:

  • Include longer blocking steps (2+ hours)

  • Add 0.1-0.3% Triton X-100 for better penetration

  • Use detergent in washing steps to reduce non-specific binding

For Immunoprecipitation:

• Improve extraction and binding:

  • Pre-clear lysates thoroughly

  • Extend antibody binding time (overnight at 4°C)

  • Optimize antibody amount (0.5-4.0 μg per 1-3 mg lysate )

  • Consider using protein A/G beads instead of agarose for certain antibodies

• Reduce non-specific binding:

  • Include additional washing steps with increasing stringency

  • Add 0.1% SDS to washing buffer for final washes

  • Block beads with BSA before adding antibody

When troubleshooting, make methodical changes to one parameter at a time while maintaining appropriate controls to identify the specific factor affecting performance.

How can researchers distinguish between specific and non-specific signals when using ADCY6 antibodies?

Distinguishing between specific and non-specific signals is crucial for accurate interpretation of ADCY6 antibody results:

Verification Strategies for Western Blotting:

• Molecular weight confirmation:

  • ADCY6 should appear at 130-150 kDa

  • Non-specific bands at dramatically different molecular weights can be identified

  • Multiple bands may represent post-translational modifications or isoforms

• Peptide competition:

  • Pre-incubate antibody with immunizing peptide

  • Specific bands should disappear while non-specific bands remain

  • For example, blocking peptides can be purchased for certain ADCY6 antibodies

• Knockdown/knockout verification:

  • Compare samples with reduced ADCY6 expression (siRNA, CRISPR)

  • Specific bands should show reduced intensity while non-specific bands remain unchanged

  • Studies have validated antibodies using Adcy6-/- tissues

Verification Strategies for Immunostaining:

• Pattern recognition:

  • ADCY6 shows specific subcellular localization patterns in different tissues

  • In inner ear hair cells, it localizes to the basal portion of stereocilia

  • Non-specific staining typically shows random or ubiquitous patterns

• Counterstaining with established markers:

  • Co-stain with markers of known ADCY6-associated structures

  • In studies of stereocilia, ADCY6 distribution was compared with ADGRV1

  • Non-overlapping patterns with established markers suggest non-specificity

• Control tissue comparisons:

  • Compare tissues with known high vs. low ADCY6 expression

  • Include tissues from knockout models when available

  • For instance, comparing wild-type vs. Adcy6-/- inner ear tissues

Quantitative Analysis Approaches:

• Signal-to-noise ratio calculations:

  • Compare intensity of target band/region to background

  • Establish minimum threshold ratios for accepting results as specific

  • Use digital image analysis tools for objective assessment

• Titration analysis:

  • Perform antibody dilution series

  • Specific signals typically decrease proportionally with dilution

  • Non-specific background may decrease at different rates

By implementing these verification strategies systematically, researchers can confidently distinguish between specific ADCY6 signals and experimental artifacts, leading to more reliable and reproducible research outcomes.

What are the latest findings on ADCY6's role in disease pathogenesis and potential therapeutic targeting?

Recent research has revealed several novel aspects of ADCY6's involvement in disease pathogenesis:

Cancer Biology:

• ADCY6 functions as a tumor suppressor in oral tongue squamous cell carcinoma (OTSCC)

• Lower ADCY6 expression correlates with poorer prognosis in OTSCC patients

• Mechanistically, ADCY6 impairs the Hippo signaling pathway to reduce malignant biological behavior in OTSCC

• Future therapeutic direction: Strategies to upregulate or restore ADCY6 expression could potentially inhibit OTSCC progression

Developmental Disorders:

• Four ADCY6 mutations have been linked to lethal arthrogryposis multiplex congenita

• These mutations affect residues positioned at critical interfaces: between AC6 C2 domain and Gαs, between AC6 C1 and C2 domains, and between AC6 and its ATP substrate

• These mutations likely impair AC6's ability to synthesize cAMP, which is crucial for muscle, joint, and nervous system development

• Future therapeutic direction: Early identification of carriers and potential gene therapy approaches

Renal Physiology:

• Collecting duct-specific knockout of ADCY6 causes a urine-concentrating defect

• ADCY6 is implicated in arginine vasopressin (AVP)-stimulated renal water reabsorption

• Future therapeutic direction: Targeting ADCY6 or its downstream pathways might offer new approaches for treating water balance disorders

Sensory System Function:

• While ADCY6 knockout mice showed normal cochlear and retinal structure and function, ADCY6 localizes to specialized structures in sensory cells

• In inner ear hair cells, ADCY6 localization depends on the ankle link complex, which is associated with Usher syndrome proteins

• Future research direction: Investigating potential roles of ADCY6 in sensory cell stress responses or age-related degenerative conditions

Emerging Therapeutic Strategies:

• Gene therapy approaches: Delivering functional ADCY6 to tissues with mutated or downregulated expression

• Small molecule modulators: Developing compounds that enhance remaining ADCY6 activity in partial loss-of-function scenarios

• Downstream pathway targeting: Identifying critical nodes in ADCY6 signaling networks as alternative therapeutic targets

As research progresses, ADCY6 antibodies will continue to play crucial roles in validating these mechanisms and evaluating potential therapeutic interventions.

How are ADCY6 antibodies being used in cutting-edge research methodologies?

ADCY6 antibodies are being incorporated into several advanced research methodologies:

Single-Cell Analysis:

• ADCY6 antibodies are being employed in advanced single-cell protein profiling techniques

• These approaches allow researchers to map ADCY6 expression heterogeneity within tissues

• Example: Analysis of renal collecting duct cells shows variability in ADCY6 expression that may impact water homeostasis regulation

Super-Resolution Microscopy:

• High-resolution localization of ADCY6 in specialized cellular structures

• In inner ear hair cells, precise localization to stereocilia bases has been demonstrated

• These techniques reveal nanoscale organization of signaling complexes containing ADCY6

Proximity-Dependent Labeling:

• BioID and APEX2-based approaches are being combined with ADCY6 antibodies

• These methods identify proximal proteins in living cells, revealing novel interaction partners

• Subsequent validation with co-immunoprecipitation using ADCY6 antibodies confirms direct interactions

Tissue Clearing and 3D Imaging:

• Whole-organ imaging with ADCY6 antibodies after tissue clearing

• Allows visualization of ADCY6 distribution across intact tissue architecture

• Particularly valuable for understanding spatial organization in complex organs like kidney and brain

Multiplexed Immunofluorescence:

• Simultaneous detection of ADCY6 alongside multiple pathway components

• Cyclic immunofluorescence methods allow visualization of 20+ proteins on the same tissue section

• Reveals colocalization patterns and pathway relationships in situ

In vivo Proximity Ligation Assay (PLA):

• Detection of protein-protein interactions involving ADCY6 in intact tissue

• Visualizes interactions that may be lost in conventional co-immunoprecipitation approaches

• Particularly valuable for studying membrane protein complexes

Functional Genomics Integration:

• Combining CRISPR screens with ADCY6 antibody-based validation

• Identifies genes that regulate ADCY6 expression, localization, or function

• Creates systems-level understanding of ADCY6 regulation

These advanced methodologies are expanding our understanding of ADCY6 biology beyond traditional approaches, revealing new insights into its functional organization and regulatory mechanisms. As antibody technologies continue to evolve, we can expect even more sophisticated applications of ADCY6 antibodies in future research.