ADCY1 Antibody

What is ADCY1 and what is its function in cellular signaling?

ADCY1 (Adenylate Cyclase 1, Brain) is a member of the Adenylyl cyclase class-4/guanylyl cyclase protein family. In humans, the canonical protein has a reported length of 1119 amino acid residues and a molecular mass of 123.4 kDa . ADCY1 is primarily localized in the cell membrane and cytoplasm, with notable expression in the zona glomerulosa and zona fasciculata of the adrenal gland .

Functionally, ADCY1 catalyzes the formation of the signaling molecule cyclic AMP (cAMP) in response to G-protein signaling . This enzymatic activity positions ADCY1 as a key component in signal transduction pathways affecting gene expression, cell proliferation, and apoptosis. Recent research has identified ADCY1 as a potential regulator of platinum-based chemotherapy sensitivity in lung cancer cells, suggesting its involvement in cell survival pathways .

What are the key structural features of ADCY1 protein that researchers should consider when selecting antibodies?

When selecting antibodies for ADCY1 detection, researchers should consider several key structural features:

The protein's substantial size (123.4 kDa) necessitates careful selection of gel percentage for Western blotting and may require special transfer conditions for larger proteins . ADCY1 contains transmembrane domains that anchor it to the cell membrane, which may affect epitope accessibility depending on sample preparation methods .

Several commercial antibodies target specific regions of ADCY1, such as amino acids 835-1061, which may have different accessibility depending on protein conformation and experimental conditions . These epitope considerations are particularly important for applications requiring native protein detection.

The protein may undergo post-translational modifications that could affect antibody binding. Although specific modifications aren't detailed in the search results, researchers should consider potential phosphorylation sites and other modifications when interpreting experimental outcomes.

What are the common applications of ADCY1 antibodies in research?

ADCY1 antibodies are versatile tools with multiple validated applications:

Western Blotting (WB): Widely used for detecting ADCY1 protein expression levels and validating antibody specificity, with recommended dilutions ranging from 0.01-2 μg/ml .

Immunohistochemistry (IHC): Used to examine ADCY1 expression patterns in tissue sections, with typical working dilutions of 5-20 μg/ml .

Immunofluorescence/Immunocytochemistry (IF/ICC): Employed to visualize subcellular localization of ADCY1, with recommended concentrations similar to IHC (5-20 μg/ml) .

Enzyme-Linked Immunosorbent Assay (ELISA): Useful for quantitative detection of ADCY1 in solution .

Immunoprecipitation (IP): Enables the isolation of ADCY1 and its binding partners for further analysis .

Each application may require specific optimization of antibody concentration, sample preparation, and detection methods to achieve optimal results.

How can ADCY1 antibodies be used to investigate its role in cancer drug resistance?

ADCY1 antibodies serve as valuable tools for investigating the emerging role of ADCY1 in cancer drug resistance through several methodological approaches:

Expression Analysis: Recent research has revealed that ADCY1 expression levels may correlate with cisplatin sensitivity in lung cancer cells. Knockdown of ADCY1 significantly decreased sensitivity to cisplatin and increased IC50 values in lung cancer cell lines (A549 and H1299) . Antibodies can be used to quantify ADCY1 protein levels in sensitive versus resistant cell lines or patient samples.

Mechanism Investigation: The search results indicate that ADCY1 may regulate cisplatin resistance by affecting cell proliferation, apoptosis, and cell cycle progression . Researchers can use ADCY1 antibodies in combination with markers for these processes (e.g., Ki-67, cleaved caspase-3, cyclins) to elucidate the underlying mechanisms.

Biomarker Development: ADCY1 polymorphism rs2293106 (c.3090G>A) has been associated with platinum-based chemotherapy effectiveness in non-small cell lung cancer patients . Antibodies that can detect specific ADCY1 variants could potentially serve as predictive biomarkers for treatment response.

Downstream Pathway Analysis: ADCY1 appears to control apoptosis by regulating the classical apoptosis biomarkers Bax and Bcl2 . Co-immunoprecipitation with ADCY1 antibodies can help identify interaction partners in the resistance pathway.

What methodological considerations are important when using ADCY1 antibodies for immunohistochemistry?

Successful immunohistochemical detection of ADCY1 requires careful attention to several methodological factors:

Antigen Retrieval: ADCY1's membrane localization may necessitate optimized antigen retrieval methods to expose epitopes masked during fixation. Heat-induced epitope retrieval (HIER) in citrate buffer (pH 6.0) or EDTA buffer (pH 9.0) should be tested systematically.

Antibody Selection: Choose antibodies validated specifically for IHC applications. Many ADCY1 antibodies are raised against specific regions (e.g., amino acids 835-1061) that may have differential accessibility in fixed tissues .

Concentration Optimization: Follow manufacturer recommendations as a starting point (typically 5-20 μg/ml for IHC as suggested by Abbexa), but always perform a titration series to determine optimal concentration for your specific tissue samples .

Control Selection: Include positive control tissues with known ADCY1 expression (adrenal gland sections showing zona glomerulosa and zona fasciculata) . Negative controls should include isotype control antibodies and tissues not expected to express ADCY1.

Specificity Validation: Consider peptide competition assays using the immunizing peptide to confirm binding specificity. Some suppliers provide recombinant ADCY1 fragments that can be used for this purpose .

Detection Systems: For potentially low-abundance proteins like ADCY1, consider amplification systems such as polymer-based detection or tyramide signal amplification to enhance sensitivity while maintaining specificity.

How can researchers differentiate between ADCY1 and other adenylate cyclase isoforms in experimental systems?

Differentiating between ADCY1 and other adenylate cyclase isoforms requires strategic approaches:

Epitope-Specific Antibodies: Select antibodies targeting unique regions of ADCY1 not conserved in other isoforms. Several commercial antibodies target specific regions of ADCY1, such as amino acids 835-1061 .

Western Blot Analysis: Different adenylate cyclase isoforms have distinct molecular weights. ADCY1's 123.4 kDa size can help differentiate it from other isoforms in Western blot analysis .

Expression Pattern Analysis: ADCY1 shows tissue-specific expression patterns, being notably expressed in the zona glomerulosa and zona fasciculata of the adrenal gland . This characteristic expression pattern can help distinguish it from other isoforms.

Knockdown/Knockout Validation: Use siRNA knockdown (as demonstrated in study ) or CRISPR-based approaches targeting ADCY1-specific sequences to confirm antibody specificity and functional outcomes.

Activation Characteristics: ADCY1 is described as "Ca(2+)/calmodulin-activated adenylyl cyclase" , suggesting a distinct regulatory mechanism that can be exploited in functional studies to differentiate from other isoforms.

Multiple Antibody Validation: Use multiple antibodies targeting different regions of ADCY1 to confirm results and increase confidence in isoform-specific detection.

What are the optimal protocols for using ADCY1 antibodies in Western blotting?

For optimal Western blot detection of ADCY1, researchers should follow these methodological guidelines:

Sample Preparation: Extract proteins using RIPA buffer supplemented with protease inhibitors. Given ADCY1's membrane localization, ensure adequate solubilization of membrane proteins .

Gel Selection: Use 8-10% SDS-PAGE gels to adequately resolve ADCY1's 123.4 kDa molecular weight . Include appropriate molecular weight markers covering the 100-150 kDa range.

Transfer Conditions: For large proteins like ADCY1, use wet transfer methods with extended transfer times (90-120 minutes at 100V or overnight at 30V, 4°C). PVDF membranes are recommended for their higher protein binding capacity.

Blocking: Block with 5% non-fat dry milk or BSA in TBST for 1 hour at room temperature to minimize non-specific binding.

Primary Antibody Incubation: Follow manufacturer recommendations for dilution; Abbexa recommends 0.01-2 μg/ml for Western blotting . Incubate overnight at 4°C with gentle rocking for optimal sensitivity.

Washing: Wash 3-4 times with TBST, 5-10 minutes each, to remove unbound antibody.

Secondary Antibody: Most ADCY1 antibodies are rabbit polyclonals, so use appropriate anti-rabbit HRP-conjugated secondary antibody . Incubate for 1 hour at room temperature.

Controls: Include positive control lysates from tissues known to express ADCY1 (adrenal tissues) and negative controls. Consider using lysates from ADCY1 knockdown experiments as specificity controls .

Signal Detection: Use ECL-based chemiluminescent detection systems. For potentially low-abundance signals, consider enhanced chemiluminescent substrates or digital imaging systems with higher sensitivity.

How should researchers troubleshoot non-specific binding when using ADCY1 antibodies?

When encountering non-specific binding with ADCY1 antibodies, follow these troubleshooting steps:

Antibody Titration: Test a range of antibody concentrations around the manufacturer's recommendation (0.01-2 μg/ml for WB, 5-20 μg/ml for IHC) . Non-specific binding often occurs with excessive antibody concentration.

Blocking Optimization: Test different blocking agents (BSA, normal serum from the secondary antibody host species, commercial blocking solutions) and increase blocking time or concentration.

Buffer Adjustments: Increase washing stringency by adding more salt (up to 500 mM NaCl) to wash buffers or include mild detergents (0.1-0.3% Triton X-100) to reduce hydrophobic interactions.

Cross-Reactivity Assessment: Consider potential cross-reactivity with other adenylate cyclase family members. Validate results using genetic approaches (siRNA knockdown) as demonstrated in study .

Antigen Retrieval Modification: For IHC/IF applications, systematically test different antigen retrieval methods, as over-retrieval can sometimes lead to increased background.

Secondary Antibody Controls: Include a no-primary antibody control to assess secondary antibody specificity. Consider changing secondary antibody supplier if background persists.

Sample Quality: Ensure samples are fresh and properly preserved. Degraded proteins can contribute to non-specific binding. Store samples according to recommendations (e.g., aliquot and store at -20°C, avoid repeated freeze/thaw cycles) .

Validation Across Applications: If non-specific binding persists in one application, validate the antibody in an alternative application (e.g., if WB shows multiple bands, test in IHC with appropriate controls).

What factors influence the selection of ADCY1 antibodies for different experimental applications?

Multiple factors should guide the selection of ADCY1 antibodies for specific experimental applications:

Application Validation: Ensure the antibody has been specifically validated for your intended application. According to search results, many ADCY1 antibodies are validated for multiple applications including WB, IHC, IF/ICC, and IP .

Epitope Considerations: For detecting specific domains or regions of ADCY1, select antibodies targeting those specific regions. Search results mention antibodies targeting amino acids 835-1061, which may be important for certain functional studies .

Species Reactivity: Verify compatibility with your experimental model. Many commercial ADCY1 antibodies react with human, mouse, and/or rat proteins , but cross-reactivity should be confirmed experimentally.

Clonality: Most available ADCY1 antibodies appear to be polyclonal , which generally offers higher sensitivity but potentially lower specificity compared to monoclonals. For highly specific detection, look for monoclonal options if available.

Format Requirements: Most available ADCY1 antibodies are unconjugated , requiring secondary detection. For direct detection methods, consider whether conjugated antibodies are available or needed.

Buffer Compatibility: Consider whether the antibody formulation is compatible with your experimental conditions. Abbexa's antibody is supplied in "0.01 M PBS, pH 7.4, containing 0.05% Proclin-300, 50% glycerol" , which may affect dilution calculations.

Validation Evidence: Review available validation data from manufacturers, including Western blots showing a single band at the expected molecular weight (123.4 kDa) and IHC/IF images demonstrating expected subcellular localization.

What controls should be included when validating ADCY1 antibodies for research applications?

Comprehensive validation of ADCY1 antibodies requires multiple controls:

Positive and Negative Tissue Controls: Include tissues with known ADCY1 expression (adrenal gland sections with zona glomerulosa and zona fasciculata) and tissues not expected to express ADCY1 as negative controls.

Molecular Weight Verification: In Western blotting, confirm that the antibody detects a band at the expected molecular weight of 123.4 kDa . Multiple bands may indicate non-specificity or post-translational modifications.

Peptide Competition Assay: Pre-incubate the antibody with excess immunizing peptide (if available from the manufacturer) to confirm binding specificity. This should eliminate specific staining.

Genetic Validation: Use tissues or cells with ADCY1 knockdown/knockout as the most stringent specificity control. Study used siRNA knockdown of ADCY1, which could serve as an excellent control system.

Isotype Controls: Include appropriate isotype control antibodies at the same concentration as the primary antibody to assess non-specific binding.

Multi-technique Validation: Validate the antibody across multiple techniques (e.g., WB, IHC, and IF) to increase confidence in specificity. Consistent results across techniques strongly support antibody specificity.

Lot-to-Lot Consistency: Test new antibody lots against previously validated lots to ensure consistency, particularly important for polyclonal antibodies which may show lot-to-lot variation.

Application-Specific Controls: Include method-specific controls (e.g., loading controls for WB, no-primary controls for IHC/IF, IgG controls for IP).

How can ADCY1 antibodies be used to study its role in cancer progression and treatment resistance?

ADCY1 antibodies offer multiple approaches to investigate its emerging role in cancer:

Expression Analysis in Clinical Samples: Research has shown that ADCY1 is overexpressed in lung squamous cell carcinoma compared to normal tissue, and higher expression correlates with poorer prognosis in lung adenocarcinoma patients . Antibodies can be used to screen patient cohorts to identify correlations between ADCY1 expression and clinical outcomes.

Mechanism Investigation: ADCY1 affects cisplatin resistance in lung cancer cells by regulating cell proliferation, apoptosis, and cell cycle progression . Researchers can use ADCY1 antibodies in combination with functional assays to elucidate the underlying mechanisms.

Signaling Pathway Analysis: As ADCY1 catalyzes cAMP formation in response to G-protein signaling , antibodies can help map the signaling networks connecting ADCY1 to downstream effectors like Bax and Bcl2 .

Therapeutic Target Validation: Given ADCY1's role in cisplatin resistance, antibodies can help validate it as a potential therapeutic target. Co-immunoprecipitation with ADCY1 antibodies can identify interaction partners that might serve as alternative targets.

Biomarker Development: The association of ADCY1 polymorphism rs2293106 with platinum-based chemotherapy efficacy suggests potential as a predictive biomarker. Antibodies that can distinguish between variants could have clinical utility.

What methodological approaches can be used to quantify ADCY1 protein levels in biological samples?

Several methodological approaches enable quantitative analysis of ADCY1:

Quantitative Western Blotting: Use standard curves of recombinant ADCY1 protein alongside samples. Employ fluorescent secondary antibodies rather than HRP-based detection for wider linear dynamic range. Include appropriate loading controls for normalization.

ELISA Development: Develop sandwich ELISA assays using capture and detection ADCY1 antibodies. This provides higher throughput than Western blotting and potentially greater sensitivity.

Mass Spectrometry: Use ADCY1 antibodies for immunoprecipitation followed by mass spectrometric analysis. This enables absolute quantification and can detect post-translational modifications.

Quantitative Immunohistochemistry: Apply digital pathology approaches to quantify ADCY1 staining in tissue sections. Measure staining intensity, percent positive cells, and H-scores for comparative analysis.

In-Cell Western Assay: For cultured cells, use in-cell Western techniques with infrared detection systems to quantify ADCY1 in fixed cells in microplate format.

Flow Cytometry: For single-cell analysis, optimize ADCY1 antibodies for flow cytometry to measure expression levels across cell populations and identify heterogeneity.

Proximity Ligation Assay: For studying ADCY1 interactions quantitatively, combine ADCY1 antibodies with antibodies against potential interaction partners in proximity ligation assays.

How does ADCY1 contribute to cisplatin resistance in lung cancer cells, and how can this be studied?

Recent research highlights ADCY1's role in cisplatin resistance with several investigative approaches:

Expression Correlation Studies: ADCY1 shows higher expression in lung cancer cells than normal cells, but lower expression in cisplatin-resistant (A549-DDP) cells compared to parental A549 cells . This inverse correlation with resistance can be studied using antibodies to quantify ADCY1 levels.

Functional Manipulation: Knockdown of ADCY1 significantly decreased sensitivity to cisplatin and increased IC50 values in lung cancer cell lines (A549 and H1299) . This approach can be extended to other cell lines and combined with antibody detection to confirm knockdown efficiency.

Molecular Mechanism Investigation: ADCY1 appears to regulate apoptotic pathways by modulating expression of Bax and Bcl2 . Co-immunostaining with ADCY1 and these apoptotic markers can reveal spatial relationships and potential regulatory mechanisms.

Genetic Association Studies: ADCY1 polymorphism rs2293106 (c.3090G>A) is associated with platinum-based chemotherapy effectiveness . Antibodies specific to variant forms could help translate genetic findings to protein-level mechanisms.

Signaling Pathway Analysis: As an adenylate cyclase, ADCY1 generates cAMP in response to G-protein signaling . Studying how this canonical pathway intersects with cisplatin resistance mechanisms could reveal novel therapeutic targets.

Clinical Sample Validation: Higher ADCY1 expression correlates with poorer prognosis in lung adenocarcinoma patients . This finding can be validated in larger patient cohorts using ADCY1 antibodies on tissue microarrays.

What are the latest findings on ADCY1's role in disease pathways beyond cancer?

While the search results focus primarily on ADCY1's role in cancer, particularly lung cancer, several aspects suggest broader disease relevance:

Genetic Associations: The mention of DFNB44 as a synonym for ADCY1 suggests potential involvement in hereditary hearing loss, as DFNB designations typically refer to autosomal recessive nonsyndromic hearing loss loci.

Neurological Functions: ADCY1's designation as "brain adenylate cyclase" points to important neurological functions that warrant investigation in neurological disorders.

Signal Transduction Implications: As a key enzyme in cAMP production , ADCY1 likely influences numerous physiological processes regulated by this second messenger, including hormone response, synaptic plasticity, and immune function.

Cross-Species Conservation: ADCY1 orthologs have been reported in mouse, rat, bovine, frog, chimpanzee, and chicken species , suggesting evolutionarily conserved functions that may extend beyond currently known roles.

Tissue-Specific Expression: The notable expression in adrenal gland tissues suggests potential roles in endocrine regulation and stress response pathways that remain to be fully explored.

How can researchers combine ADCY1 antibodies with other techniques to gain comprehensive insights into its function?

Integrating multiple methodological approaches provides more comprehensive understanding of ADCY1 function:

Multi-omics Integration: Combine protein-level detection using ADCY1 antibodies with transcriptomic and genomic data. Study used RNA sequencing to identify downstream genes affected by ADCY1 that may be associated with drug resistance.

Live-Cell Imaging: Pair fixed-cell antibody staining with live-cell imaging using fluorescent cAMP sensors to correlate ADCY1 expression with functional activity in real time.

Protein-Protein Interaction Mapping: Use ADCY1 antibodies for co-immunoprecipitation followed by mass spectrometry to identify novel interaction partners. This approach can reveal new components of ADCY1-mediated signaling networks.

Structure-Function Analysis: Combine immunological detection of ADCY1 variants with functional assays measuring adenylyl cyclase activity to correlate structural features with enzymatic function.

CRISPR Genome Editing: Extend beyond siRNA knockdown (as used in study ) to create stable ADCY1 knockout or knock-in cell lines, validating antibody specificity while enabling long-term functional studies.

Patient-Derived Models: Apply ADCY1 antibodies to patient-derived xenografts or organoids to study its expression and function in more clinically relevant models than established cell lines.

Computational Modeling: Integrate antibody-based protein quantification data into computational models of G-protein signaling pathways to predict system-level effects of ADCY1 modulation.