ANO8 Antibody

What is ANO8 and why is it important in calcium signaling research?

ANO8 belongs to the anoctamin family and functions as a key tether in the formation of endoplasmic reticulum (ER) and plasma membrane (PM) junctions. It plays an essential role in STIM1-STIM1 interaction and STIM1-Orai1 interaction at ER/PM PI(4,5)P₂-rich compartments. ANO8 is significant because it assembles core calcium signaling proteins (Orai1, PMCA, STIM1, IP₃ receptors, and SERCA2) at the ER/PM junctions, thereby mediating Orai1 channel inactivation and controlling receptor-stimulated Ca²⁺ signaling and oscillations . Detecting and studying ANO8 is crucial for understanding fundamental calcium signaling mechanisms in cellular physiology.

What applications are ANO8 antibodies validated for in research settings?

Based on available research materials, ANO8 antibodies have been validated for multiple experimental applications:

The antibodies show reactivity with human, mouse, and rat samples, making them versatile tools for comparative studies across species .

How do you determine the optimal antibody concentration for ANO8 detection in different experimental systems?

For optimal ANO8 detection, a titration approach is recommended rather than relying solely on manufacturer-suggested dilutions. Begin with a dilution series (e.g., 1:100, 1:250, 1:500, 1:1000) in your specific system. For IHC applications, start with the recommended range of 1:250-1:1000 . Include appropriate positive controls (such as mouse brain tissue) and negative controls (omitting primary antibody or using tissue known to lack ANO8 expression).

Evaluate signal-to-noise ratio across different concentrations to determine the optimal dilution that provides specific staining with minimal background. This is particularly important for ANO8 detection because its expression levels can vary significantly between tissues and experimental conditions. Remember that optimal concentration may need adjustment based on:

• Sample preparation method

• Fixation protocol

• Antigen retrieval technique (TE buffer pH 9.0 is suggested for ANO8 IHC, with citrate buffer pH 6.0 as an alternative)

• Detection system sensitivity

What are the recommended sample preparation methods for detecting ANO8 in different subcellular compartments?

When studying ANO8 as an ER/PM junction tether, sample preparation requires careful consideration to preserve these delicate membrane structures:

For immunofluorescence/confocal microscopy:

• Fixation: 4% paraformaldehyde (10-15 minutes at room temperature) preserves membrane architecture while allowing antibody access.

• Permeabilization: Use gentle detergents (0.1% Triton X-100 or 0.1% saponin) to avoid disrupting membrane junctions.

• Blocking: 5% BSA or 10% serum from the secondary antibody host species (1 hour at room temperature).

• Primary antibody: Apply ANO8 antibody at optimized dilution overnight at 4°C.

• Secondary antibody: Fluorophore-conjugated antibody (1:500-1:1000) for 1-2 hours at room temperature.

For electron microscopy (EM) of ER/PM junctions containing ANO8:
EM has been successfully used to visualize ANO8-enriched junctions, showing that ANO8 increases both the number and size of ER/PM junctions . Standard EM fixation protocols with glutaraldehyde followed by osmium tetroxide provide good preservation of these structures.

For subcellular fractionation:
When biochemically isolating ER/PM junctions, use sucrose gradient ultracentrifugation methods optimized for membrane contact sites, followed by immunoblotting for ANO8 detection.

How can TIRF microscopy be optimized for studying ANO8 localization at ER/PM junctions?

Total Internal Reflection Fluorescence (TIRF) microscopy is an ideal technique for studying ANO8 at ER/PM junctions due to its ability to visualize events near the plasma membrane with high resolution. Based on published methodologies:

• Sample preparation:

  • Express fluorescently tagged constructs (ANO8-YFP, mCherry-STIM1, Orai1-CFP) in appropriate cell lines

  • For fixed samples, use minimal fixation (2% PFA for 10 minutes) to preserve fluorescent proteins

  • Mount samples in imaging chambers with glass bottoms of appropriate thickness for TIRF

• TIRF setup optimization:

  • Adjust the incident angle to achieve an evanescent field depth of ~100-150 nm

  • Use multi-color TIRF to simultaneously visualize ANO8 with interaction partners

  • For dynamic studies, maintain cells at 37°C with appropriate buffers

• Analysis strategies:

  • Quantify puncta formation before and after store depletion

  • Measure co-localization coefficients between ANO8 and STIM1/Orai1

  • Perform TIRF-Z scanning as described in research to visualize the spatial relationship between ANO8 puncta and STIM1 clusters

Research has shown that ANO8 forms puncta at the TIRF field that increase after store depletion, with N-terminally tagged mCherry-STIM1 clusters forming at ANO8 puncta but in a plane farther from the plasma membrane than ANO8 .

What controls should be included when using ANO8 antibodies for protein interaction studies?

When designing co-immunoprecipitation (Co-IP) or FRET experiments to study ANO8 interactions with calcium signaling proteins, the following controls are essential:

For Co-IP studies:

• Input control: Analyze 5-10% of pre-IP lysate to confirm protein expression

• Negative controls:

  • IgG control: Use matched isotype control antibody to assess non-specific binding

  • Knockdown control: Include samples with ANO8 siRNA/shRNA to demonstrate specificity

• Reciprocal IP: Perform reverse IP (e.g., IP with STIM1 antibody, blot for ANO8)

• Stimulus controls: Compare resting cells vs. store-depleted conditions

For FRET experiments:

• Donor-only control: Cells expressing only the donor fluorophore

• Acceptor-only control: Cells expressing only the acceptor fluorophore

• Negative interaction control: Non-interacting proteins with the same fluorophores

• Positive control: Known interaction partners labeled with the same fluorophores

Published research demonstrates significant basal FRET between STIM1-CFP and ANO8-YFP that was enhanced by expression of HA-Orai1, while minimal basal FRET was observed between Orai1 and ANO8 even when co-expressed with STIM1 .

How does ANO8 knockdown affect STIM1-Orai1 clustering and what methods detect this change?

ANO8 knockdown significantly impairs the formation of STIM1 puncta and STIM1-Orai1 complexes at ER/PM junctions, with several complementary methods demonstrating this effect:

Functional impact:

• 70% reduction in Orai1 current without altering channel inward rectification

• 50% reduction in native store-operated Ca²⁺ influx

• 50% reduction in STIM1 puncta at the TIRF plane

Detection methods:

• Electrophysiology: Patch-clamp recordings show decreased current density in ANO8 knockdown cells

• Calcium imaging: Reduced store-operated Ca²⁺ entry measured by fluorescent indicators

• TIRF microscopy: Quantitative analysis of STIM1 and Orai1 puncta formation

• Co-IP assays: Reduced native STIM1-Orai1 interaction following store depletion

• Surface biotinylation: Decreased PM-localized Orai1 and STIM1 at junctions

These methods collectively demonstrate that ANO8 functions as a bona fide ER/PM tether that regulates the assembly and interaction of STIM1 and Orai1 at junctions, thereby controlling Orai1 activation by STIM1 and the duration of Ca²⁺ influx .

What experimental approaches can distinguish between ANO8's role in STIM1-STIM1 interaction versus its tethering function?

This question addresses a sophisticated aspect of ANO8 function that requires specialized experimental approaches to delineate:

To isolate effects on STIM1-STIM1 interaction:

• FRET between STIM1 molecules: Measure STIM1-STIM1 FRET efficiency in the ER (away from PM) with and without ANO8

• Co-IP of differently tagged STIM1 proteins: Assess STIM1-STIM1 interaction in cells with ANO8 expression or knockdown

• BiFC (Bimolecular Fluorescence Complementation): Split fluorescent protein fragments attached to STIM1 molecules will fluoresce only upon STIM1-STIM1 interaction

To isolate effects on tethering function:

• Constitutively active STIM1 mutants: Use pre-clustered STIM1 mutants (STIM1(D76A), STIM1-Kras, STIM1(ΔCTID)) whose clustering is independent of store depletion

  • ANO8 knockdown reduced clustering of these mutants and Orai1 current density, indicating a direct effect on ER/PM junctions

• Electron microscopy: Direct visualization and quantification of ER/PM junctions

  • ANO8 expression increased both size and number of junctions across multiple EM sections

• Surface biotinylation assays: Measure PM-localized Orai1 and associated STIM1

  • ANO8 increased PM Orai1 levels and junction-associated STIM1, even in resting state

These approaches collectively demonstrate that ANO8 has dual functions: enhancing STIM1-STIM1 interaction in the ER and increasing the formation of ER/PM junctions where STIM1-Orai1 complexes assemble.

How can researchers distinguish between SARAF-dependent and ANO8-mediated forms of Orai1 current inactivation?

• Electrophysiological approaches:

  • Compare SCDI kinetics with 3mM EGTA (slower Ca²⁺ buffer) vs. 10mM BAPTA (faster Ca²⁺ buffer)

  • Measure inactivation in SARAF knockdown cells with and without ANO8 expression

  • Analyze current traces for distinctive kinetic components

• Molecular manipulation experiments:

  • SARAF knockdown/knockout with ANO8 expression shows only partial reduction in SCDI

  • Compare effects of ANO8 on wild-type Orai1 vs. inactivation-resistant Orai1 mutants

  • Express ANO8 mutants lacking specific protein interaction domains

• Proximity analysis techniques:

  • FRET measurements between ANO8, SARAF, STIM1, and Orai1

  • Co-IP experiments to detect molecular complexes under different conditions

Research has shown that ANO8 increases interaction of SARAF with STIM1 (enhancing SARAF-dependent SCDI) but also reveals a SARAF-independent form of Orai1 inactivation . This novel form involves ANO8 facilitating SERCA2-mediated Ca²⁺ influx into the ER, creating a distinct regulatory mechanism for calcium signaling.

How can researchers address epitope masking issues when ANO8 forms complexes with calcium signaling proteins?

Epitope masking occurs when protein-protein interactions obscure antibody binding sites. For ANO8, which forms complexes with multiple calcium signaling proteins (STIM1, Orai1, SERCA2, IP₃R), this presents a significant challenge:

Detection strategies to overcome masking:

• Multiple antibody approach: Use antibodies targeting different epitopes of ANO8

  • Compare results from commercial antibodies with different immunogen sequences

  • The antibody targeting sequence "REHDSGGREEARAEGSGLDPATSSEKASAKAKGSTAGGHGPERPKRPGSLLAPNNVMKLKQIIPLQGKFLSSGAT" may access different regions than peptide-based antibodies

• Sample preparation modifications:

  • Test different detergents in lysis buffers (CHAPS vs. Triton X-100 vs. digitonin)

  • Try partial protein denaturation protocols that maintain epitope structure

  • Use cross-linking approaches to stabilize complexes before detection

• Alternative detection methods:

  • Proximity ligation assay (PLA) to detect ANO8 in close proximity to partners

  • Mass spectrometry-based approaches for complex composition analysis

  • Expression of epitope-tagged ANO8 constructs with tags in different protein regions

Documentation and validation:

• Always document experimental conditions where epitope masking might occur

• Compare results across resting and stimulated conditions

• Validate findings with orthogonal approaches (e.g., fluorescent protein fusion imaging)

What strategies can resolve inconsistent ANO8 antibody results between different experimental systems?

When researchers encounter variable results with ANO8 antibodies across different cell types or experimental conditions, systematic troubleshooting is essential:

Source of variability and resolution strategies:

• Expression level differences:

  • Quantify ANO8 expression by qPCR across systems

  • Use Western blot with recombinant protein standards for absolute quantification

  • Adjust antibody concentration based on expression level in each system

• Post-translational modifications:

  • ANO8 may undergo different modifications affecting epitope recognition

  • Test antibodies specific for unmodified ANO8

  • Compare results with phosphatase-treated samples

• Isoform variation:

  • Check for tissue-specific ANO8 isoforms in your system

  • Design experiments to detect specific isoforms (e.g., isoform-specific primers)

  • Cross-reference with UniProt IDs (Q9HCE9 human, Q6PB70 mouse)

• Methodology standardization:

  • Implement consistent sample preparation across systems

  • For IHC, standardize antigen retrieval (TE buffer pH 9.0 recommended)

  • Use automated systems where possible to reduce technical variation

Reconciliation of data:
Create a detailed comparison table documenting all variables across experimental systems including fixation, permeabilization, blocking, antibody concentration, and detection method. This systematic approach will identify the source of variability and guide protocol optimization.

How should researchers interpret data from ANO8 knockdown experiments in the context of calcium signaling studies?

ANO8 knockdown experiments have revealed its critical role in calcium signaling, but interpreting such data requires careful consideration of several factors:

Interpretation framework:

• Direct vs. indirect effects:

  • ANO8 knockdown reduces STIM1-STIM1 and STIM1-Orai1 interaction

  • It decreases ER/PM junctions, Orai1 current, and Ca²⁺ influx

  • Consider whether observed phenotypes result from direct ANO8 loss or secondary effects

• Compensatory mechanisms:

  • Other anoctamin family members may partially compensate

  • Prolonged knockdown may trigger different adaptations than acute depletion

  • Compare siRNA (acute) vs. shRNA/CRISPR (chronic) approaches

• System-specific considerations:

  • Effects may vary between cell types with different calcium signaling machinery

  • Primary cells vs. cell lines may show distinct responses

  • Consider the relative importance of ANO8 vs. other tethers in your system

Analytical approaches:

• Time-course analysis: Examine effects at different time points after knockdown

• Dose-dependent studies: Use partial knockdown to reveal threshold effects

• Rescue experiments: Re-express ANO8 or specific domains to identify critical regions

• Combined knockdowns: Target ANO8 together with other tethers to assess redundancy

Interpretation of calcium signaling data:
Research shows that ANO8 knockdown reduces store-operated Ca²⁺ entry by approximately 50% and Orai1 current by 70% . This partial rather than complete inhibition suggests that other tethering mechanisms contribute to ER/PM junction formation and calcium signaling, even in the absence of ANO8.

What are the most promising approaches for studying ANO8's role in tissue-specific calcium signaling contexts?

Understanding ANO8's function across different physiological contexts represents a frontier in calcium signaling research:

Advanced methodological approaches:

• Tissue-specific conditional knockout models:

  • Generate ANO8 floxed mice for tissue-specific deletion

  • Compare phenotypes across tissues with different calcium signaling requirements

  • Assess compensatory changes in other tethering proteins

• Advanced imaging techniques:

  • Super-resolution microscopy (STORM/PALM) to visualize ANO8 nanoscale organization

  • Lattice light-sheet microscopy for 3D dynamics in living tissues

  • Correlative light and electron microscopy (CLEM) to link ANO8 location with ultrastructure

• Physiological context exploration:

  • Examine ANO8 in specialized calcium signaling contexts (neurons, immune cells, etc.)

  • Study its role during development and aging

  • Investigate ANO8 function in disease models with altered calcium homeostasis

Current antibodies show reactivity with human, mouse, and rat samples , providing tools for comparative studies across species. The documented expression in mouse brain tissue suggests neuronal calcium signaling as a promising area for investigation.

How can researchers differentiate between ANO8's potential calcium channel function versus its tethering role?

ANO8 belongs to the anoctamin family, members of which can function as calcium-activated chloride channels, yet current research emphasizes its tethering function:

Experimental strategies to distinguish these roles:

• Structure-function analysis:

  • Generate ANO8 mutants that preserve tethering but disrupt potential channel function

  • Use patch-clamp electrophysiology to directly assess channel activity

  • Compare ANO8 to established anoctamin channels (ANO1/2) in parallel assays

• Ion flux measurements:

  • Use chloride-sensitive fluorescent indicators to detect potential ANO8-mediated flux

  • Perform ion substitution experiments to determine ion selectivity

  • Examine calcium dependence of any observed channel activity

• Molecular-level approaches:

  • Compare ANO8 structure with known channel-forming anoctamins

  • Identify and mutate potential pore-forming regions

  • Assess oligomerization properties characteristic of ion channels

Current literature states that ANO8 "may act as a calcium-activated chloride channel" , suggesting this function remains hypothetical. In contrast, its tethering role in ER/PM junctions has been experimentally demonstrated . Resolving this dual functionality question would significantly advance our understanding of ANO8 biology.