ADCY10 Antibody

What is ADCY10 and why is it important in research?

ADCY10, also known as soluble adenylyl cyclase (sAC), represents a distinct class of mammalian adenylyl cyclase that significantly differs from transmembrane adenylyl cyclases. Unlike other adenylyl cyclases except ADCY9, ADCY10 is insensitive to forskolin stimulation and is uniquely modulated by calcium and bicarbonate ions . ADCY10 is primarily localized in the cytoplasm and functions as a general bicarbonate sensor throughout the body . Research has demonstrated its critical role in the generation of cAMP in spermatozoa, suggesting important functions in sperm maturation through the epididymis, capacitation, hypermotility, and possibly the acrosome reaction . Understanding ADCY10's unique regulatory properties provides insight into specialized cAMP signaling mechanisms that operate independently of G-protein coupled pathways.

The importance of ADCY10 in research extends beyond reproductive biology, as its bicarbonate-sensing capabilities implicate it in various physiological processes requiring pH or metabolic sensing. Its distinct regulatory properties make it an intriguing target for studying alternative cAMP generation pathways in cellular signaling networks across multiple tissues and organ systems . Researchers frequently utilize ADCY10 antibodies to investigate these functions in various experimental contexts.

What types of ADCY10 antibodies are available for research applications?

Several types of ADCY10 antibodies are available for research applications, primarily dominated by rabbit polyclonal antibodies targeting different epitopes of the protein. Researchers can select antibodies directed against specific regions of ADCY10, including the N-terminal region and various amino acid sequences (AA 1101-1200, AA 557-583, AA 396-445) . These region-specific antibodies allow for targeted investigations of ADCY10 domains and their potential functional significance.

The most commonly available ADCY10 antibodies are polyclonal antibodies raised in rabbits, which offer broad epitope recognition . These antibodies vary in their conjugation status, with most being unconjugated, though some biotinylated versions exist for specialized applications . The reactivity profile of these antibodies spans multiple species, with many products demonstrating cross-reactivity with human, rat, mouse, rabbit, cow, dog, guinea pig, and horse ADCY10 . This cross-species reactivity enables comparative studies across model organisms, though researchers should verify the predicted reactivity percentages (typically ranging from 93-100% depending on species) for their specific experimental system .

How can I validate the specificity of an ADCY10 antibody for my experimental system?

Validating ADCY10 antibody specificity is a critical step before conducting extensive experiments. Begin with western blotting using cell lysates known to express ADCY10, as this technique allows visualization of the antibody's binding pattern . Based on published research, ADCY10 should appear as a band with an apparent molecular mass of approximately 48 kDa, though some tissue sources may show an additional 34 kDa band . The absence of expected bands or the presence of numerous non-specific bands may indicate poor antibody specificity.

Include appropriate positive and negative controls in your validation process. Positive controls might include cell lines known to express ADCY10, such as MLTC-1 cells which have been confirmed to express ADCY10 through both western blotting and immunofluorescence . For negative controls, consider using cells where ADCY10 expression has been knocked down through siRNA or CRISPR-Cas9, or simply omit the primary antibody in your staining protocol . Additionally, peptide competition assays, where the antibody is pre-incubated with the immunizing peptide, can confirm binding specificity by demonstrating signal reduction. The comprehensive validation should also include immunocytochemistry to confirm the expected cytoplasmic localization pattern of ADCY10, as confirmed in studies showing ADCY10 distributed throughout the cytoplasm in MLTC-1 cells .

What are the optimal conditions for using ADCY10 antibodies in Western blotting?

Optimizing western blotting conditions for ADCY10 detection requires careful consideration of several parameters. Based on published protocols, the expected molecular weight of ADCY10 is approximately 48 kDa, though in some tissues a second band at 34 kDa may be observed . When preparing samples, use complete protease inhibitor cocktails in your lysis buffer to prevent degradation of ADCY10, which can affect band patterns and signal intensity.

For electrophoresis, 10-12% polyacrylamide gels generally provide optimal resolution in the 30-50 kDa range where ADCY10 is detected. Following transfer to membranes, blocking with 5% non-fat dry milk in TBST (Tris-buffered saline with 0.1% Tween-20) for 1 hour at room temperature helps reduce non-specific binding . For primary antibody incubation, a dilution range of 1:500 to 1:2000 is typically effective, though this should be optimized for each specific antibody . Overnight incubation at 4°C generally yields the best signal-to-noise ratio. For signal detection, both chemiluminescence and fluorescence-based methods are suitable, with the choice depending on your laboratory's equipment and sensitivity requirements. Including positive controls, such as cell lysates confirmed to express ADCY10 (e.g., MLTC-1 cells), is crucial for validating your results .

How can I effectively use ADCY10 antibodies for immunofluorescence and immunohistochemistry?

For successful immunofluorescence and immunohistochemistry applications with ADCY10 antibodies, proper sample preparation and optimization of staining conditions are essential. When fixing cells or tissues, 4% paraformaldehyde for 15-20 minutes at room temperature generally preserves ADCY10 antigenicity while maintaining cellular architecture. For tissue sections, antigen retrieval may be necessary, with citrate buffer (pH 6.0) being commonly effective for ADCY10 detection.

During the staining procedure, permeabilization with 0.1-0.3% Triton X-100 ensures antibody access to the cytoplasmic ADCY10, followed by blocking with 5-10% normal serum from the same species as the secondary antibody . Primary antibody incubation should be performed at dilutions ranging from 1:100 to 1:500, with overnight incubation at 4°C often providing optimal results . For visualization, fluorophore-conjugated secondary antibodies that match your microscopy setup should be selected. When interpreting results, expect ADCY10 staining throughout the cytoplasm, as confirmed by published immunofluorescence studies . Including a nuclear counterstain (such as DAPI) helps visualize cellular architecture and confirm subcellular localization. As with all immunostaining procedures, negative controls (omitting primary antibody) are essential to confirm staining specificity and should always be run in parallel with your experimental samples .

What considerations are important when selecting an ADCY10 antibody for specific research applications?

Selecting the appropriate ADCY10 antibody requires consideration of multiple factors tailored to your specific research objectives. First, identify the target species you're working with and confirm antibody cross-reactivity through predicted reactivity data. Available ADCY10 antibodies demonstrate various reactivity patterns across species including human, rat, mouse, rabbit, cow, dog, guinea pig, and horse, with reactivity percentages typically ranging from 93-100% .

Second, consider the epitope recognition region based on your research questions. If you're investigating domain-specific functions or interactions, select antibodies targeting specific regions like the N-terminal domain or particular amino acid sequences (e.g., AA 396-445, AA 557-583, or AA 1101-1200) . The epitope location may affect antibody performance in different applications – for instance, some epitopes may be masked in certain experimental conditions or in protein complexes.

Third, match the antibody's validated applications with your experimental techniques. Different ADCY10 antibodies are validated for specific applications such as Western blotting (WB), ELISA, immunofluorescence (IF), immunohistochemistry (IHC), and flow cytometry (FACS) . Finally, consider antibody format and modifications (unconjugated vs. conjugated) based on your detection systems. Most ADCY10 antibodies are available unconjugated, though biotinylated versions exist for specialized detection methods . Thorough validation of your selected antibody in your specific experimental system is essential before proceeding with large-scale experiments.

How can ADCY10 antibodies be used to study its interaction with signaling pathways involving cAMP?

Investigating ADCY10's role in cAMP signaling pathways requires sophisticated experimental approaches utilizing ADCY10 antibodies. Co-immunoprecipitation (Co-IP) experiments represent a powerful method to identify proteins that physically interact with ADCY10 within signaling complexes. By using ADCY10 antibodies to pull down the protein from cell lysates, researchers can identify novel binding partners through subsequent mass spectrometry or western blotting with antibodies against suspected interacting proteins.

Proximity ligation assays (PLA) offer another advanced approach to visualize protein-protein interactions in situ. This technique uses ADCY10 antibodies in combination with antibodies against potential interacting proteins to generate fluorescent signals only when the proteins are in close proximity (<40 nm). For investigating dynamic signaling events, researchers can combine ADCY10 immunocytochemistry with phospho-specific antibodies against downstream effectors of cAMP signaling (such as PKA substrates) following stimulation with ADCY10 activators like bicarbonate. This approach has been successfully employed in studies examining the relationship between LH stimulation, ADCY10 activity, and subsequent cAMP production in MLTC-1 cells . Additionally, ADCY10 antibodies can be used in chromatin immunoprecipitation (ChIP) experiments to investigate whether ADCY10 associates with transcriptional complexes, potentially linking cAMP signaling directly to transcriptional regulation.

What methods can be used to distinguish between ADCY10 and transmembrane adenylyl cyclases in experimental systems?

Distinguishing between ADCY10 (soluble adenylyl cyclase) and transmembrane adenylyl cyclases (tmACs) requires strategic experimental approaches that exploit their differential regulatory mechanisms. Immunological approaches using ADCY10-specific antibodies in immunofluorescence or immunohistochemistry can visualize the distinct subcellular localization patterns – ADCY10 exhibits cytoplasmic distribution, while tmACs localize to the plasma membrane . This visual distinction provides initial evidence for differential localization of these cyclase types.

Pharmacological approaches offer powerful tools for functional discrimination. ADCY10 is uniquely inhibited by specific compounds including KH7, LRE1, 2-CE, and 4-CE, while being insensitive to forskolin, which activates most tmACs . Conversely, tmACs can be selectively inhibited by P-site inhibitors such as 2',5'-dideoxyadenosine (ddAdo5') at appropriate concentrations . In experimental designs, researchers can use these differential inhibitor sensitivities to parse the relative contributions of ADCY10 versus tmACs to observed cAMP responses. Research has demonstrated that in MLTC-1 cells, KH7 at 25 μM completely abolishes LH-stimulated cAMP accumulation, while ddAdo5' requires a much higher concentration (250 μM) to achieve similar inhibition . Additionally, genetic approaches using siRNA or CRISPR-Cas9 to specifically knockdown ADCY10 expression, verified by western blotting with ADCY10 antibodies, can further confirm the specific contribution of ADCY10 to observed phenotypes.

How can researchers investigate ADCY10's role in bicarbonate sensing using antibody-based approaches?

Investigating ADCY10's function as a bicarbonate sensor requires sophisticated experimental designs incorporating antibody-based approaches with functional assays. Immunofluorescence using ADCY10 antibodies can track potential subcellular redistribution of ADCY10 in response to bicarbonate exposure, which may reveal regulatory mechanisms through compartmentalization. Time-course experiments capturing protein localization at various intervals after bicarbonate treatment can provide insights into the dynamics of this response.

Co-immunoprecipitation experiments using ADCY10 antibodies before and after bicarbonate stimulation can identify changes in protein-protein interaction networks that may regulate ADCY10's bicarbonate-sensing capabilities. These studies might reveal bicarbonate-dependent binding partners that modulate ADCY10 activity. For functional investigations, researchers can combine ADCY10 immunocytochemistry with real-time cAMP measurements using FRET-based sensors or luciferase-based reporter systems, as demonstrated in studies with MLTC-1 cells transiently expressing cyclic AMP-responsive luciferase . This approach allows correlation between ADCY10 expression levels (quantified by immunostaining) and functional responses to bicarbonate.

Mutational analysis of ADCY10's bicarbonate-binding domains, verified by antibody-based expression confirmation, can provide mechanistic insights into the structural basis of bicarbonate sensing. Site-directed mutagenesis of suspected bicarbonate-interacting residues, followed by immunoblotting to confirm expression and functional assays to assess bicarbonate responsiveness, creates a powerful approach to map critical regulatory sites. Additionally, domain-specific antibodies targeting different regions of ADCY10 may be used to investigate which structural components undergo conformational changes upon bicarbonate binding.

How should researchers address inconsistent results when using ADCY10 antibodies in different experimental contexts?

Inconsistent results with ADCY10 antibodies across different experimental contexts require systematic troubleshooting approaches. First, evaluate antibody quality and storage conditions, as antibody degradation can significantly impact performance. ADCY10 antibodies should typically be stored according to manufacturer recommendations, usually at -20°C with minimal freeze-thaw cycles . Prepare small working aliquots to avoid repeated freezing and thawing of the entire stock.

Second, optimize protein extraction methods based on ADCY10's cytoplasmic localization. Standard lysis buffers containing 1% NP-40 or Triton X-100 with protease inhibitors are generally suitable, but extraction efficiency may vary across tissue types . For tissues with high protease activity, increasing protease inhibitor concentrations may preserve ADCY10 integrity. Third, adjust antibody concentration and incubation conditions for each specific application. What works optimally for western blotting may not be ideal for immunohistochemistry or immunoprecipitation. Perform antibody titration experiments to determine optimal concentrations for each application.

Fourth, validate results using multiple methodologies. If western blotting yields inconsistent results, confirm findings using immunofluorescence or functional assays with ADCY10 inhibitors like KH7 or LRE1 . Finally, account for potential biological variability in ADCY10 expression across different cell types, developmental stages, or physiological conditions. Research has shown that ADCY10 can manifest as single or double bands (48 kDa and sometimes 34 kDa) depending on the tissue source, which may reflect tissue-specific post-translational modifications or isoforms .

What controls are essential when using ADCY10 antibodies in quantitative research applications?

Implementing rigorous controls is crucial for generating reliable quantitative data with ADCY10 antibodies. Positive controls should include samples known to express ADCY10, such as MLTC-1 cells which have been verified to express ADCY10 through multiple methods . For tissue-specific studies, include tissues with documented ADCY10 expression as positive controls. Negative controls should involve samples where ADCY10 expression is absent or substantially reduced, which could include ADCY10 knockout models or cells treated with verified ADCY10-targeting siRNAs.

Technical controls for immunoblotting must include loading controls appropriate for your experimental context. While housekeeping proteins like β-actin or GAPDH are commonly used, verify that your experimental treatments do not alter their expression. For immunostaining, include secondary-only controls (omitting primary antibody) to assess non-specific binding of secondary antibodies . Additionally, peptide competition controls, where the antibody is pre-incubated with the immunizing peptide before staining, can confirm signal specificity.

For quantitative comparisons across multiple samples or conditions, standardize all experimental parameters including sample preparation, antibody concentrations, incubation times, and image acquisition settings. When performing densitometric analysis of western blots, ensure signals fall within the linear range of detection to avoid saturation, which compromises quantification accuracy. For immunofluorescence quantification, use standardized exposure settings across all samples and include fluorescence intensity calibration standards when possible. Finally, validate antibody-based quantification with orthogonal methods such as qPCR for mRNA levels or functional assays measuring ADCY10 activity.

How can researchers differentiate between specific and non-specific signals when using ADCY10 antibodies?

Differentiating between specific and non-specific signals requires systematic validation approaches when working with ADCY10 antibodies. First, verify band specificity in western blotting by confirming that observed bands match the expected molecular weight of ADCY10 (primarily 48 kDa, with a possible 34 kDa band in some tissues) . Multiple bands at unexpected molecular weights may indicate non-specific binding or protein degradation. Peptide competition experiments, where the primary antibody is pre-incubated with excess immunizing peptide, should specifically reduce or eliminate the ADCY10 signal while non-specific signals typically remain unaffected.

For immunostaining applications, compare the observed staining pattern with the established subcellular localization of ADCY10, which should appear predominantly in the cytoplasm . Staining patterns that deviate significantly from this expected distribution may represent non-specific binding. Perform parallel staining with two different ADCY10 antibodies targeting distinct epitopes – concordant staining patterns strongly suggest specificity, while discordant patterns raise concerns about non-specific binding.

Genetic approaches provide definitive validation strategies. Reduced or absent signal in samples where ADCY10 has been knocked down via siRNA or CRISPR-Cas9 provides compelling evidence for antibody specificity. Similarly, increased signal intensity in samples overexpressing ADCY10 further confirms specificity. For functional validation, correlate antibody-detected ADCY10 levels with functional outcomes using specific inhibitors like KH7, LRE1, 2-CE, or 4-CE . Samples with higher ADCY10 expression (as detected by the antibody) should demonstrate greater sensitivity to these inhibitors if the antibody is truly detecting ADCY10.

How are ADCY10 antibodies being used in research on reproductive biology and fertility?

ADCY10 antibodies have become instrumental in reproductive biology research, particularly in studies investigating male fertility mechanisms. Researchers utilize these antibodies to examine ADCY10 expression and localization in spermatozoa at different maturation stages through immunofluorescence and immunohistochemistry techniques . These approaches have revealed ADCY10's dynamic expression patterns during sperm transit through the epididymis and its correlation with acquiring fertilization competence.

In functional studies, ADCY10 antibodies enable researchers to correlate protein expression levels with sperm parameters including motility, capacitation, and acrosome reaction capacity. Immunoblotting with ADCY10 antibodies, combined with functional assays using ADCY10 inhibitors like KH7, has demonstrated the critical role of this enzyme in cAMP-dependent signaling pathways that regulate sperm function . In male infertility research, ADCY10 antibodies facilitate comparative studies between normal and pathological samples, potentially identifying alterations in ADCY10 expression or localization associated with specific infertility phenotypes.

Beyond male reproduction, ADCY10 antibodies are being employed to investigate potential roles in female reproductive tissues. Studies examining ADCY10 expression in ovarian cells, particularly in response to gonadotropin stimulation, provide insights into potential functions in folliculogenesis and oocyte maturation. The combined approach of ADCY10 immunodetection with functional manipulation using specific inhibitors has revealed unexpected signaling pathways in reproductive tissues, such as the contribution of ADCY10 to LH-stimulated steroidogenesis in Leydig cells .

What technological advancements are improving the sensitivity and specificity of ADCY10 antibody-based detection methods?

Recent technological advances have significantly enhanced the capabilities of ADCY10 antibody-based detection methods. Super-resolution microscopy techniques, including Structured Illumination Microscopy (SIM) and Stochastic Optical Reconstruction Microscopy (STORM), now enable visualization of ADCY10 distribution at nanoscale resolution. These approaches reveal detailed subcellular localization patterns that conventional microscopy cannot resolve, potentially identifying specialized compartmentalization of ADCY10 that correlates with functional domains.

Multiplexed immunofluorescence technologies allow simultaneous detection of ADCY10 alongside multiple other proteins of interest. These methods utilize antibodies labeled with spectrally distinct fluorophores or employ sequential staining with antibody stripping between rounds, enabling comprehensive analysis of ADCY10's relationship with interacting proteins and signaling components within the same sample. This approach provides powerful contextual information about signaling networks rather than isolated pathway components.

Mass spectrometry-based proteomics approaches coupled with ADCY10 immunoprecipitation have dramatically improved sensitivity for detecting low-abundance interaction partners. Techniques like proximity-dependent biotin identification (BioID) or APEX labeling, followed by streptavidin pulldown and mass spectrometry, enable mapping of the ADCY10 protein interaction network in living cells under physiological conditions. Additionally, advanced computational tools for image analysis have enhanced quantitative evaluation of ADCY10 immunostaining. Machine learning algorithms can now accurately segment cellular compartments and quantify staining intensity across large datasets, enabling high-throughput analysis of ADCY10 expression and localization under various experimental conditions.

How might ADCY10 antibodies contribute to understanding pathological conditions beyond reproductive disorders?

ADCY10 antibodies are increasingly recognized as valuable tools for investigating pathological conditions extending beyond reproductive disorders. In metabolic research, ADCY10 antibodies help examine how this bicarbonate-sensing enzyme participates in energy metabolism and cellular responses to metabolic stress. Immunohistochemical studies in pancreatic tissues have revealed ADCY10 expression patterns that suggest potential roles in insulin secretion and glucose homeostasis, opening avenues for diabetes research.

In neurological disorders research, ADCY10 antibodies facilitate investigation of its potential functions in neuronal signaling and brain metabolism. The unique regulation of ADCY10 by bicarbonate may connect it to brain pH homeostasis, which is disrupted in conditions like ischemia, seizures, and neurodegenerative diseases. Immunostaining of brain tissue sections from disease models and patient samples can reveal alterations in ADCY10 expression or localization that may contribute to pathological processes.

Cancer research represents another emerging application, as cAMP signaling pathways are frequently dysregulated in various malignancies. ADCY10 antibodies enable comparative studies between normal and tumor tissues, potentially identifying altered expression patterns that correlate with tumor progression or therapeutic response. The unique regulatory properties of ADCY10, distinct from transmembrane adenylyl cyclases, may offer novel therapeutic targeting strategies that could be monitored using antibody-based detection methods. These diverse applications highlight the expanding utility of ADCY10 antibodies beyond their traditional use in reproductive biology, positioning them as valuable tools for understanding fundamental signaling mechanisms across multiple physiological and pathological contexts.