ADCY10 Antibody, FITC conjugated

What is ADCY10 and why is it significant in biological research?

ADCY10, also known as soluble adenylyl cyclase (sAC), is an enzyme that catalyzes the formation of the critical signaling molecule cyclic AMP (cAMP). Unlike transmembrane adenylyl cyclases, ADCY10 functions as a soluble protein that can be distributed throughout various cellular compartments including the cytoplasm, mitochondria, nucleus, and cell projections such as cilia. The significance of ADCY10 lies in its role as a sensor that mediates responses to changes in cellular bicarbonate and CO₂ levels, making it critical for multiple physiological processes. ADCY10 plays essential roles in mammalian spermatogenesis, sperm capacitation, and ciliary beat regulation, making it a valuable target for reproductive biology, respiratory research, and broader cell signaling studies .

What are the common applications for ADCY10 antibodies in research?

ADCY10 antibodies are primarily used in several experimental applications including western blotting (WB), flow cytometry (FC), immunohistochemistry on paraffin-embedded tissues (IHC-P), enzyme-linked immunosorbent assay (ELISA), and various immunofluorescence (IF) techniques. These applications allow researchers to detect ADCY10 expression patterns, localize the protein within tissues or subcellular compartments, and quantify expression levels. For FITC-conjugated variants specifically, the most common applications are flow cytometry and direct immunofluorescence microscopy, which leverage the fluorescent properties of FITC for visualization without requiring secondary antibody steps .

What species reactivity can I expect from commercially available ADCY10 antibodies?

The species reactivity of ADCY10 antibodies varies by product and manufacturer. Based on the available information, many ADCY10 antibodies show reactivity to human samples, with cross-reactivity to rat and mouse commonly reported. Some antibodies demonstrate broader cross-reactivity to additional species including rabbit, cow, dog, guinea pig, and horse. This cross-reactivity is due to the high conservation of ADCY10 across mammalian species, with predicted reactivity percentages typically ranging from 93-100% for various mammals. When selecting an ADCY10 antibody for your research, it's essential to verify the specific species reactivity required for your experimental system .

What are the key differences between using FITC-conjugated versus unconjugated ADCY10 antibodies?

FITC-conjugated ADCY10 antibodies offer several methodological advantages over unconjugated versions, particularly for flow cytometry and direct immunofluorescence. The primary difference is that FITC-conjugated antibodies eliminate the need for secondary antibody incubation steps, reducing experimental time, decreasing background signal, and minimizing cross-reactivity issues. FITC (fluorescein isothiocyanate) conjugation provides direct visualization with excitation/emission wavelengths of approximately 495/519 nm, appearing green when visualized.

How should I optimize fixation protocols for ADCY10 immunodetection in different subcellular locations?

Optimizing fixation protocols is crucial since ADCY10 exhibits diverse subcellular localization including cell membrane, cytoskeleton, perinuclear region, nucleus, cilia, and mitochondria. For preserving ADCY10 in these various locations:

• Membrane and cytoskeletal ADCY10: A combination of 2-4% paraformaldehyde with 0.1-0.2% glutaraldehyde can maintain membrane associations while preserving protein antigenicity.

• Nuclear and perinuclear ADCY10: 4% paraformaldehyde alone for 15-20 minutes at room temperature often provides optimal nuclear epitope preservation.

• Mitochondrial ADCY10: Methanol fixation (-20°C for 10 minutes) can provide superior results for mitochondrial protein detection, though this should be validated empirically.

• Ciliary ADCY10: For ciliary structures, careful fixation with 2% paraformaldehyde for 10 minutes followed by methanol post-fixation often preserves both structure and antigenicity.

The choice of fixation protocol should be validated experimentally for your specific ADCY10 antibody, as the ADCY10 epitope recognition (N-terminal, central region, or C-terminal) will influence fixation sensitivity .

What dilution ranges are recommended for FITC-conjugated ADCY10 antibodies in different applications?

Optimal dilution ranges for FITC-conjugated ADCY10 antibodies vary by application:

• Flow cytometry: The recommended dilution range is typically 1:10 to 1:50, with optimization needed based on cell type and expression level. This relatively concentrated application is necessary due to the direct detection without signal amplification.

• Immunofluorescence microscopy: Starting dilutions of 1:50 to 1:200 are often suitable, with optimization based on tissue type and fixation method.

• Western blotting: If using FITC-conjugated antibodies for direct fluorescence detection in Western blotting (less common), higher concentrations (1:100 to 1:500) may be necessary for detection systems with appropriate excitation/emission capabilities.

These recommendations serve as starting points, and titration experiments should be performed to determine optimal signal-to-noise ratios for specific experimental conditions. The limited photostability of FITC means that greater antibody concentrations may be needed compared to more stable fluorophores .

How can I distinguish between different isoforms of ADCY10 using antibody-based approaches?

The single ADCY10 gene gives rise to multiple isoforms with distinct characteristics and subcellular distributions. When designing experiments to distinguish between these isoforms:

• Epitope selection: Choose antibodies targeting unique regions of specific isoforms. For example, antibodies targeting the N-terminal region (aa 1-100) will detect different isoform subsets compared to those targeting the central catalytic domain (aa 557-583) or C-terminal region (aa 1100-1200).

• Combined immunoprecipitation and Western blotting: Use antibodies recognizing different epitopes to immunoprecipitate ADCY10, followed by Western blotting with isoform-specific antibodies to distinguish between variants based on molecular weight (full-length ADCY10 is approximately 187 kDa).

• Subcellular fractionation: Combine cellular fractionation techniques with immunoblotting to distinguish isoforms based on their compartmentalization (membrane, cytosolic, nuclear, or mitochondrial).

• siRNA knockdown validation: Confirm isoform specificity by employing siRNAs targeting specific exons, then validating antibody detection patterns after knockdown.

For greatest accuracy, complementary techniques like RT-PCR or mass spectrometry should be used alongside antibody-based methods to confirm isoform identification .

What strategies can resolve cross-reactivity issues when using ADCY10 antibodies in multi-protein detection systems?

When using ADCY10 antibodies in multiplex detection systems, several strategies can minimize cross-reactivity:

• Sequential staining protocols: For multiply-labeled immunofluorescence, complete one antibody staining cycle (primary and secondary) with thorough washing before beginning the next. This prevents cross-reaction between detection systems.

• Absorption controls: Pre-absorb the ADCY10 antibody with recombinant protein containing the immunizing peptide sequence to eliminate non-specific binding.

• Isotype-matched controls: Use isotype-matched control antibodies (rabbit IgG for polyclonal ADCY10 antibodies) to establish background staining levels.

• Species-specific secondary antibodies: When using multiple primary antibodies, select those raised in different host species and use highly cross-absorbed secondary antibodies.

• Spectral unmixing: For FITC-conjugated antibodies used alongside other fluorophores, employ spectral unmixing algorithms during image acquisition to separate overlapping emission spectra.

• Antibody fragmentation: In some cases, using F(ab) or F(ab')2 fragments of ADCY10 antibodies can reduce non-specific binding through Fc receptors.

Validating specificity through knockout/knockdown controls remains the gold standard for confirming antibody specificity regardless of the approach used .

How can I optimize detection of phosphorylation-dependent changes in ADCY10 activity?

ADCY10 activity is regulated through various post-translational modifications, including phosphorylation. To optimize detection of phosphorylation-dependent changes:

• Phosphatase inhibitors: Include comprehensive phosphatase inhibitor cocktails during sample preparation to preserve phosphorylation states (e.g., 10 mM sodium fluoride, 2 mM sodium orthovanadate, 1 mM β-glycerophosphate).

• Phosphorylation-state specific antibodies: Where available, use antibodies that specifically recognize phosphorylated forms of ADCY10.

• Phos-tag™ SDS-PAGE: Incorporate Phos-tag™ in polyacrylamide gels to separate phosphorylated from non-phosphorylated ADCY10 based on mobility shift.

• Functional activity assays: Couple immunoprecipitation of ADCY10 with enzymatic activity assays measuring cAMP production to correlate phosphorylation status with functional changes.

• Kinase/phosphatase treatments: Treat samples with specific kinases or phosphatases before immunodetection to confirm phosphorylation-dependent antibody recognition.

• Proximity ligation assays: For FITC-conjugated ADCY10 antibodies, combine with antibodies against known interacting phosphoproteins using proximity ligation techniques to visualize phosphorylation-dependent protein interactions.

These approaches can help researchers dissect the complex regulation of ADCY10 through post-translational modifications that affect its catalytic activity .

What are the recommended controls for validating ADCY10 antibody specificity in immunofluorescence studies?

Rigorous validation of ADCY10 antibody specificity requires multiple complementary controls:

• Peptide competition: Pre-incubate the ADCY10 antibody with excess immunizing peptide before staining to confirm binding specificity.

• Genetic validation: Use ADCY10 knockout or knockdown samples alongside wildtype to demonstrate signal reduction/elimination.

• Multiple antibody validation: Compare staining patterns using multiple ADCY10 antibodies targeting different epitopes (N-terminal, central, C-terminal).

• Recombinant protein controls: Include cells overexpressing tagged ADCY10 to confirm co-localization between tag antibodies and ADCY10 antibodies.

• Secondary-only controls: Omit primary antibody but include all other staining steps to establish background fluorescence.

• Isotype controls: Use matched isotype control antibodies at equivalent concentrations to identify non-specific binding.

• Cross-reactivity assessment: Test the antibody on tissues/cells known to be negative for ADCY10 expression.

• Excitation/emission controls: For FITC-conjugated antibodies specifically, include controls to account for potential autofluorescence in the FITC channel, particularly in fixed tissues.

Implementing these controls is essential for publication-quality research and ensures reliable interpretation of ADCY10 localization studies .

How should I troubleshoot weak or absent ADCY10 signal in Western blotting applications?

When encountering weak or absent ADCY10 signal in Western blotting, consider the following systematic troubleshooting approach:

• Sample preparation:

  • Ensure complete protease inhibition during lysis

  • Avoid excessive sample heating which may denature large proteins like ADCY10 (187 kDa)

  • Use freshly prepared samples when possible

• Protein loading and transfer:

  • Increase protein loading (50-100 μg may be necessary)

  • Use gradient gels (4-12%) to better resolve high molecular weight proteins

  • Extend transfer time (overnight at low voltage) for large proteins

  • Consider semi-dry transfer systems with specialty buffers for large proteins

• Antibody conditions:

  • Try different antibody dilutions (1:500 to 1:2000 range)

  • Extend primary antibody incubation (overnight at 4°C)

  • For FITC-conjugated antibodies, ensure detection system compatibility with the fluorophore

  • Consider non-conjugated antibodies with signal amplification for increased sensitivity

• Blocking optimization:

  • Test alternative blocking agents (BSA vs. milk)

  • Reduce blocking time if epitope recognition is affected

• Detection considerations:

  • Use enhanced chemiluminescence substrates designed for high sensitivity

  • For fluorescent detection systems, ensure appropriate filter sets and sensitivity settings

• Expression verification:

  • Confirm ADCY10 expression in your sample type through RT-PCR or other methods

  • Consider enrichment through immunoprecipitation before Western blotting

If using FITC-conjugated antibodies directly for Western blot, remember their reduced sensitivity compared to enzyme-conjugated secondary antibody systems .

What methodological strategies can improve detection of ADCY10 in ciliary structures?

Detecting ADCY10 in ciliary structures presents unique challenges due to their small size and specialized nature. Recommended methodological strategies include:

• Super-resolution microscopy techniques:

  • Structured illumination microscopy (SIM)

  • Stimulated emission depletion microscopy (STED)

  • Photoactivated localization microscopy (PALM)
    These techniques overcome the diffraction limit of conventional microscopy, enabling visualization of ADCY10 within small ciliary structures.

• Sample preparation optimization:

  • Use gentle fixation methods (2% PFA for 10 minutes)

  • Consider methanol post-fixation for enhanced ciliary epitope exposure

  • Optimize permeabilization conditions (0.1-0.2% Triton X-100 for 5-10 minutes)

• Co-staining strategies:

  • Include established ciliary markers (acetylated tubulin, ARL13B) for co-localization

  • Use serial confocal sections with Z-stack reconstruction to fully capture ciliary structures

• Signal enhancement methods:

  • Tyramide signal amplification for non-conjugated antibodies

  • For FITC-conjugated antibodies, optimize imaging parameters to enhance detection while minimizing photobleaching

• Cell culture considerations:

  • Culture cells to confluence to promote ciliogenesis

  • Serum starvation (24-48 hours) to enhance primary cilium formation

  • Consider specialized ciliated cell models (e.g., human nasal epithelial cells, ependymal cells)

These approaches can significantly improve detection of ADCY10 in ciliary structures, where the protein plays a critical role in regulating ciliary beat through cAMP signaling .

How can FITC-conjugated ADCY10 antibodies be utilized in live-cell imaging experiments?

Live-cell imaging with FITC-conjugated ADCY10 antibodies requires specialized approaches to maintain cell viability while achieving sufficient antibody internalization:

• Membrane-permeable antibody techniques:

  • Antibody loading via microinjection for single-cell analysis

  • Protein transfection reagents (e.g., Chariot, BioPORTER) to deliver antibodies into living cells

  • Electroporation-mediated antibody delivery for difficult-to-transfect cell types

• Optimization for FITC characteristics:

  • Minimize exposure time and light intensity to reduce photobleaching

  • Buffer systems maintaining pH 7.4-8.0 for optimal FITC fluorescence

  • Antifade agents compatible with live cells (e.g., OxyFluor, ProLong Live)

• Experimental design considerations:

  • Short imaging windows (1-4 hours) to minimize antibody degradation

  • Temperature-controlled staging (37°C) to maintain physiological conditions

  • CO₂ buffering systems for experiments exceeding 30 minutes

  • Sequential rather than continuous image acquisition to reduce phototoxicity

• Validation approaches:

  • Parallel experiments with GFP-tagged ADCY10 to confirm antibody specificity

  • Cell viability assessments throughout the imaging period

  • Functional assays to ensure cellular processes remain unperturbed by antibody binding

These techniques allow researchers to track dynamic changes in ADCY10 localization in response to stimuli such as bicarbonate/CO₂ level changes or other physiological modulators .

What are the considerations for using ADCY10 antibodies in studying bicarbonate/CO₂ sensing mechanisms?

ADCY10 functions as a bicarbonate/CO₂ sensor, making antibody-based approaches valuable for studying these sensing mechanisms. Key considerations include:

• Experimental system design:

  • Controlled CO₂/bicarbonate manipulation systems (perfusion chambers, controlled atmosphere incubators)

  • Real-time monitoring capabilities for both ADCY10 localization/activity and bicarbonate/CO₂ levels

  • Simultaneous pH monitoring to distinguish direct bicarbonate effects from pH-mediated effects

• Functional correlation strategies:

  • Combine antibody-based localization with FRET-based cAMP sensors to correlate ADCY10 activity with cAMP production

  • Phosphorylation-state specific antibodies to detect activation-dependent post-translational modifications

  • Co-immunoprecipitation studies to identify dynamic interaction partners under varying bicarbonate conditions

• Tissue/cell-specific considerations:

  • Selection of appropriate cell models (sperm, ciliated epithelial cells, neurons) with established bicarbonate responsiveness

  • Specialized media formulations allowing precise bicarbonate manipulation

  • Comparative studies across tissues with different ADCY10 isoform expression

• Technical challenges with FITC-conjugated antibodies:

  • pH sensitivity of FITC fluorescence requiring careful experimental design when modulating bicarbonate/CO₂

  • Potential intensity changes with pH independent of actual ADCY10 concentration changes

  • Implementation of ratiometric approaches or pH-insensitive fluorophores for quantitative studies

These considerations enable researchers to effectively study ADCY10's role in sensing and responding to physiologically relevant changes in cellular bicarbonate and CO₂ levels .

How can multi-parametric flow cytometry be optimized for analyzing ADCY10 in heterogeneous cell populations?

Multi-parametric flow cytometry using FITC-conjugated ADCY10 antibodies requires careful panel design and optimization:

• Panel design considerations:

  • Position FITC in appropriate detector channel considering its brightness and spectral overlap

  • Complement with markers for cell identification (lineage markers), activation state, and functional parameters

  • Include markers to distinguish subcellular compartments (mitochondrial markers, nuclear markers) when permeabilization is used

  • Reserve bright fluorochromes for low abundance targets

• Sample preparation optimization:

  • Fixation/permeabilization protocol selection based on ADCY10 subcellular localization of interest

  • Titration of all antibodies individually before combined use

  • Sequential staining for surface markers before permeabilization for intracellular ADCY10 detection

  • Washing buffer optimization to reduce background and non-specific binding

• Instrument setup and controls:

  • Fluorescence-minus-one (FMO) controls including FITC-conjugated isotype control

  • Compensation controls using single-stained samples or beads

  • Standardized voltage settings for longitudinal studies

  • Daily quality control using quantitative beads

• Analysis strategies:

  • Gating hierarchies isolating specific cell populations before ADCY10 analysis

  • Quantification using median fluorescence intensity rather than percent positive

  • Correlation analyses between ADCY10 levels and functional parameters

  • tSNE or UMAP dimensionality reduction for visualizing complex relationships in heterogeneous populations

This approach enables detection of differential ADCY10 expression or activation across diverse cell types within complex samples such as peripheral blood, tissue digests, or heterogeneous culture systems .

How might advances in antibody engineering improve ADCY10 detection specificity and sensitivity?

Emerging antibody technologies offer promising avenues for enhanced ADCY10 detection:

• Recombinant antibody technologies:

  • Single-chain variable fragments (scFvs) for improved tissue penetration

  • Bi-specific antibodies targeting ADCY10 and its interaction partners simultaneously

  • Intrabodies designed for specific subcellular compartment targeting

  • Nanobodies with superior access to sterically hindered epitopes

• Advanced fluorophore conjugation:

  • Site-specific conjugation strategies ensuring consistent fluorophore:antibody ratios

  • Self-quenching antibodies that fluoresce only upon target binding

  • Photoactivatable fluorophores for super-resolution applications

  • Quantum dots with superior brightness and photostability compared to FITC

• Affinity maturation approaches:

  • Directed evolution technologies to enhance ADCY10 epitope recognition

  • Computational antibody design targeting highly specific ADCY10 regions

  • CDR grafting to improve affinity while maintaining specificity

• Functional reporting capabilities:

  • Proximity-reporting antibody pairs that generate signal only when bound to adjacent epitopes

  • Conformation-specific antibodies distinguishing active vs. inactive ADCY10

  • Integration with CRISPR/Cas systems for simultaneous visualization and manipulation

These advances promise to overcome current limitations in ADCY10 detection by providing enhanced specificity for particular isoforms, improved signal-to-noise ratios, and the ability to distinguish functional states of the protein in complex biological systems .

What considerations are important when designing experiments to study ADCY10 interactions with other signaling pathway components?

Studying ADCY10's interactions with other signaling components requires careful experimental design:

• Spatial and temporal resolution:

  • Subcellular compartment-specific isolation techniques to capture localized interactions

  • Time-course studies capturing rapid signaling dynamics

  • Live-cell imaging approaches with sufficient temporal resolution to detect transient interactions

• Proximity-based detection methods:

  • Proximity ligation assay (PLA) to visualize protein-protein interactions in situ

  • FRET/BRET approaches for real-time interaction monitoring

  • BioID or APEX2 proximity labeling to identify interaction networks

  • Co-immunoprecipitation with carefully preserved protein complexes

• Functional validation approaches:

  • Correlation of protein interactions with cAMP production measurements

  • Pathway inhibitor studies to establish causality in interaction networks

  • Mutational analysis of binding interfaces to confirm direct interactions

  • Reconstitution experiments in cell-free systems

• Specificity controls:

  • Competitive binding assays with recombinant protein fragments

  • Peptide displacement strategies targeting known interaction domains

  • Comparison across tissues with differential expression of interaction partners

  • Isoform-specific approaches to distinguish interaction patterns

• Technical considerations for FITC-conjugated antibodies:

  • Fluorophore compatibility with other detection systems in multiplexed approaches

  • Photobleaching management in long-duration experiments

  • Signal amplification strategies for detecting low-abundance complexes

These considerations enable researchers to effectively characterize ADCY10's diverse interactions across signaling networks, including its roles in cAMP-dependent pathways, bicarbonate sensing, and ciliary function regulation .

What are the current consensus best practices for ADCY10 antibody validation prior to experimental use?

Current consensus best practices for ADCY10 antibody validation include a multi-faceted approach:

• Genetic validation:

  • Testing in ADCY10 knockout/knockdown systems via CRISPR-Cas9 or RNAi

  • Heterologous expression systems with controlled ADCY10 expression

  • Isoform-specific validation using splice variant-targeted approaches

• Biochemical validation:

  • Western blotting to confirm appropriate molecular weight detection (187 kDa for full-length)

  • Immunoprecipitation followed by mass spectrometry confirmation

  • Peptide competition assays with immunizing peptide

  • Multiple antibody concordance testing using antibodies targeting different epitopes

• Application-specific validation:

  • Validation in each specific application (WB, IF, FC, IHC) independently

  • Cross-validation with orthogonal methods (mRNA expression, activity assays)

  • Tissue-specific validation across samples with known differential expression

  • Species cross-reactivity confirmation for comparative studies

• Documentation requirements:

  • Complete antibody information (clone, lot, dilution, incubation parameters)

  • Detailed sample preparation methodology

  • Imaging or detection parameters specified for reproducibility

  • Inclusion of all validation data in supplementary materials

Following these best practices ensures reliable and reproducible results when using ADCY10 antibodies for research applications, addressing concerns about antibody specificity that have affected many research fields .

How should researchers interpret discrepancies between ADCY10 antibody-based detection and functional activity measurements?

When encountering discrepancies between ADCY10 antibody detection and functional measurements, consider these interpretive frameworks:

• Potential mechanistic explanations:

  • Post-translational modifications affecting activity but not antibody recognition

  • Protein-protein interactions masking antibody epitopes without affecting activity

  • Conformational changes altering function without changing detectability

  • Subcellular compartmentalization differences affecting functional readouts

• Technical considerations:

  • Epitope specificity of the antibody relative to functional domains

  • Sensitivity differences between antibody detection and activity assays

  • Temporal dynamics differences between protein presence and activation state

  • Sample preparation differences affecting protein integrity or activation state

• Reconciliation strategies:

  • Use multiple antibodies targeting different ADCY10 epitopes

  • Employ complementary techniques (activity assays, mRNA quantification)

  • Conduct time-course studies to capture temporal dynamics

  • Perform subcellular fractionation to address compartmentalization questions

• Experimental design adjustments:

  • Include appropriate positive and negative controls for both antibody detection and activity assays

  • Standardize sample handling procedures between detection and activity measurements

  • Consider dose-response relationships rather than single measurements

  • Develop correlation analyses accounting for potential non-linear relationships