ANN4 Antibody

What is ANO4 and what is its significance in cellular physiology?

ANO4, also known as TMEM16D, is a member of the Anoctamin family of proteins recognized for their calcium-activated chloride channel activity. This protein plays a crucial role in chloride ion transport and regulation of cellular excitability across various tissue types . The significance of ANO4 extends to multiple physiological processes, particularly those involving ion channel function, which makes it relevant to research on conditions such as cystic fibrosis, epilepsy, and cardiac arrhythmias .

Understanding ANO4 function contributes to our knowledge of ion channel regulation mechanisms and may inform the development of therapeutic strategies for treating ion channel-related disorders. Research using ANO4 antibodies allows for the detection and analysis of ANO4 expression patterns in different cell types and tissues, providing critical insights into its distribution and potential functional roles.

How does the ANO4 antibody differ from other antibodies targeting ion channels?

The ANO4 polyclonal antibody (such as PACO35890) is specifically designed to target the Anoctamin-4 protein, distinguishing it from antibodies targeting other ion channels or even other members of the Anoctamin family. This antibody is typically produced in rabbits using recombinant human Anoctamin-4 protein (specifically amino acids 1-200) as the immunogen .

Unlike antibodies targeting voltage-gated channels or ligand-gated channels, the ANO4 antibody focuses on calcium-activated chloride channels, which are regulated by intracellular calcium levels rather than membrane potential changes or extracellular ligand binding. This specificity makes the ANO4 antibody particularly valuable for studying calcium-dependent chloride transport mechanisms. Additionally, when compared to other ion channel antibodies, the ANO4 antibody's application profile is optimized for techniques including Western blot, immunohistochemistry, and immunofluorescence in human samples .

What are the recommended applications and dilutions for the ANO4 antibody?

The ANO4 polyclonal antibody has been validated for multiple research applications, each requiring specific dilution ranges for optimal results:

The antibody has demonstrated high reactivity with human samples and has been validated in specific tissues including lung cancer tissue, adrenal gland tissue, and HeLa cells . For optimal results, researchers should perform preliminary tests to determine the ideal dilution for their specific experimental conditions, considering factors such as the expression level of ANO4 in their samples and the detection method employed.

How can researchers validate the specificity of ANO4 antibody binding in experimental systems?

Validating ANO4 antibody specificity is critical for ensuring experimental reliability. Researchers should employ multiple complementary approaches:

• Comparative Analysis with Multiple Antibodies: Use at least two different ANO4 antibodies targeting distinct epitopes to confirm staining patterns. Consistent localization patterns across different antibodies provide evidence of specificity.

• Genetic Controls: Implement knockout/knockdown systems (CRISPR-Cas9, siRNA) to create ANO4-deficient control samples. The antibody should show significantly reduced or absent signal in these samples compared to wild-type controls.

• Blocking Peptide Competition: Pre-incubate the ANO4 antibody with excess immunizing peptide (recombinant ANO4 protein fragments, such as the 1-200AA sequence used for PACO35890) . Diminished staining in peptide-blocked samples versus unblocked controls confirms epitope-specific binding.

• Western Blot Molecular Weight Verification: Confirm that the detected protein band corresponds to the predicted molecular weight of ANO4. This can be coupled with mass spectrometry analysis of the immunoprecipitated protein to confirm identity.

• Cross-Reactivity Assessment: Test the antibody against related proteins (other Anoctamin family members) to ensure it doesn't cross-react with homologous proteins, particularly important given the structural similarities within the Anoctamin family.

These validation steps should be performed in cell types known to express ANO4 alongside negative control tissues or cells.

What are the optimal fixation and antigen retrieval protocols for ANO4 antibody in immunohistochemistry?

Based on successful applications with the PACO35890 antibody, the following protocols have yielded reliable results when detecting ANO4 in paraffin-embedded tissues:

Fixation Protocol:

• Fix tissue samples in 10% neutral buffered formalin for 24-48 hours at room temperature

• Process tissues through graded alcohols and xylene

• Embed in paraffin maintaining temperature below 60°C to prevent protein denaturation

Antigen Retrieval Protocol:

• Section tissues at 4-6 μm thickness

• Heat-induced epitope retrieval (HIER) in citrate buffer (pH 6.0) is recommended

• Bring buffer to boiling point, then maintain at 95-98°C for 15-20 minutes

• Allow sections to cool slowly to room temperature (approximately 20 minutes)

• Wash sections in PBS (pH 7.4) three times for 5 minutes each

Immunostaining Protocol:

• Block endogenous peroxidase activity with 3% hydrogen peroxide for 10 minutes

• Block non-specific binding with 5% normal goat serum for 1 hour

• Apply PACO35890 antibody at 1:100 dilution and incubate overnight at 4°C

• Wash thoroughly in PBS-T (PBS + 0.1% Tween-20)

• Apply appropriate secondary antibody system

• Develop with DAB or other suitable chromogen

• Counterstain with hematoxylin

This protocol has been successfully applied to human lung cancer and adrenal gland tissues , but may require optimization for different tissue types or fixation conditions.

How does the expression pattern of ANO4 compare across different tissue types and disease states?

ANO4 expression shows distinct patterns across different tissues and pathological conditions, which researchers should consider when designing experiments:

Normal Tissue Distribution:

• ANO4 is expressed in multiple tissues, with notable expression in neuronal tissues, adrenal glands, and lung tissue

• Within cells, ANO4 typically localizes to plasma membranes and intracellular compartments associated with calcium signaling

• Expression levels vary by tissue type, with some tissues showing cell type-specific expression patterns

Disease-Associated Expression Changes:

• In lung cancer tissues, immunohistochemistry with ANO4 antibodies has revealed altered expression patterns compared to normal lung tissue

• Similar investigations in adrenal gland tissue have demonstrated the utility of ANO4 antibodies for characterizing expression in both normal and pathological states

Experimental Considerations:

• When studying disease-associated expression changes, researchers should include matched normal controls whenever possible

• Quantitative analysis techniques such as digital image analysis of IHC staining intensity or Western blot densitometry are recommended for comparing expression levels

• Multi-parameter analysis combining ANO4 with cell type-specific markers can provide insights into cell-specific expression changes in heterogeneous tissues

Researchers studying ANO4 should establish baseline expression levels in their specific experimental systems before examining disease-associated changes.

What strategies can be employed to optimize ANO4 antibody performance in immunofluorescence studies?

Optimizing immunofluorescence (IF) protocols for ANO4 detection requires careful attention to several factors:

Sample Preparation Optimization:

• Fixation Method Comparison: Test both paraformaldehyde (PFA) and methanol fixation, as membrane proteins like ANO4 may be detected differently depending on the fixation method

• Permeabilization Adjustment: For intracellular epitopes, compare different permeabilization agents (Triton X-100, saponin, digitonin) at varying concentrations (0.1-0.5%) and incubation times

• Blocking Solution Selection: Test different blocking agents (BSA, normal serum, commercial blocking solutions) to minimize background while preserving specific signal

Antibody Application Optimization:

• Titration Series: Perform a dilution series from 1:50 to 1:200 to determine optimal signal-to-noise ratio

• Incubation Conditions: Compare room temperature (1-2 hours) versus 4°C overnight incubation

• Secondary Antibody Selection: Use highly cross-adsorbed secondary antibodies to minimize non-specific binding

• Signal Amplification: For low-abundance targets, consider implementing tyramide signal amplification systems

Advanced Visualization Techniques:

• Co-localization Studies: Pair ANO4 antibody with organelle markers (ER, Golgi, plasma membrane) using spectrally distinct fluorophores

• Super-resolution Microscopy: For detailed subcellular localization, apply techniques such as STED or STORM

• Live-Cell Labeling: For surface epitopes, consider non-permeabilized live-cell labeling with directly conjugated antibody fragments

These optimizations have been successfully applied in HeLa cells using the PACO35890 antibody at 1:100 dilution with Alexa Fluor 488-conjugated secondary antibodies , but should be customized for each cell type under investigation.

How can researchers accurately quantify ANO4 expression levels in heterogeneous tissue samples?

Accurate quantification of ANO4 expression in complex tissue samples requires rigorous methodological approaches:

Tissue-Level Quantification Methods:

• Digital Pathology Approach:

  • Whole slide scanning of immunostained sections

  • Application of machine learning algorithms for automated tissue segmentation

  • Quantification of staining intensity across different tissue compartments

  • Statistical comparison across sample groups with appropriate normalization

• Laser Capture Microdissection:

  • Selective isolation of specific cell populations from heterogeneous samples

  • RNA extraction for qRT-PCR quantification of ANO4 transcript levels

  • Protein extraction for Western blot analysis with ANO4 antibody

  • Comparison of protein vs. transcript levels to assess post-transcriptional regulation

Single-Cell Resolution Approaches:

• Multiplex Immunofluorescence:

  • Simultaneous staining for ANO4 and cell-type-specific markers

  • High-content imaging analysis with single-cell segmentation

  • Quantification of ANO4 signal intensity in different cell populations

  • Construction of expression distribution histograms for each cell type

• Flow Cytometry-Based Quantification:

  • Preparation of single-cell suspensions from tissue samples

  • Surface and intracellular staining protocols for ANO4 detection

  • Multi-parameter analysis with lineage markers

  • Absolute quantification using calibration beads

For reliable quantification, researchers should implement appropriate normalizations to account for technical variations and include proper positive and negative controls in each experiment.

What considerations should be taken into account when using ANO4 antibodies for co-immunoprecipitation experiments?

Co-immunoprecipitation (Co-IP) with ANO4 antibodies requires careful experimental design to preserve protein-protein interactions while ensuring specificity:

Lysate Preparation Considerations:

• Detergent Selection: Use mild non-ionic detergents (0.5-1% NP-40, 0.5% digitonin, or 1% CHAPS) that preserve membrane protein complexes

• Buffer Composition: Include physiologically relevant calcium concentrations (100-300 nM) to maintain calcium-dependent interactions

• Protease/Phosphatase Inhibitors: Use comprehensive inhibitor cocktails to prevent degradation and preserve post-translational modifications

• Cross-linking Option: Consider reversible cross-linking (DSP or formaldehyde) to stabilize transient interactions

Immunoprecipitation Protocol Optimization:

• Antibody Immobilization: Compare direct coupling to beads versus indirect capture with Protein G

• Pre-clearing Step: Implement stringent pre-clearing with non-immune IgG to reduce non-specific binding

• Antibody Concentration: Titrate antibody amounts (1-5 μg per mg of lysate) to determine optimal capture efficiency

• Washing Stringency: Test graduated washing conditions to balance between preserving interactions and reducing background

Validation and Controls:

• Reciprocal Co-IP: Confirm interactions by immunoprecipitating with antibodies against suspected interaction partners

• Competition Controls: Include immunizing peptide competition controls to verify specificity

• Isotype Controls: Use matched concentration of rabbit IgG as negative control

• Input Controls: Always include input samples (5-10%) for normalization

Detection Strategies:

• Western Blotting: Use highly specific antibodies against suspected interaction partners

• Mass Spectrometry: For unbiased discovery of novel interaction partners, consider LC-MS/MS analysis

For ANO4 co-IP experiments, researchers should be particularly attentive to detergent selection, as inappropriate detergents can disrupt the native conformation of membrane proteins like ANO4.

How can ANO4 antibodies be employed to investigate the functional relationship between ANO4 and calcium signaling pathways?

ANO4 antibodies can be instrumental in elucidating the relationship between ANO4 and calcium signaling through multiple experimental approaches:

Co-localization with Calcium Signaling Components:

• Dual Immunofluorescence: Combine ANO4 antibody (1:50-1:200 dilution) with antibodies against calcium signaling components (calcium channels, pumps, or calcium-binding proteins)

• Proximity Ligation Assay (PLA): Apply PLA to detect close proximity (<40 nm) between ANO4 and calcium signaling molecules, suggesting functional interaction

• Subcellular Fractionation: Use ANO4 antibodies in Western blotting of isolated subcellular compartments associated with calcium signaling (ER, plasma membrane, calcium microdomains)

Functional Coupling Analysis:

• Calcium Imaging Combined with Immunocytochemistry:

  • Perform live calcium imaging with indicators like Fluo-4 or GCaMP

  • Fix cells immediately after recording

  • Stain with ANO4 antibody to correlate calcium responses with ANO4 expression

  • Quantify correlation between calcium transient magnitude and ANO4 expression level

• Electrophysiology with Post-Recording Immunostaining:

  • Record chloride currents in single cells

  • Mark recorded cells with intracellular dyes

  • Process for immunostaining with ANO4 antibody

  • Correlate current properties with ANO4 expression levels

Regulation of ANO4 by Calcium Signaling:

• Calcium Manipulation Experiments:

  • Treat cells with calcium ionophores or SERCA inhibitors

  • Fix and immunostain for ANO4 using optimal antibody dilution (1:100 suggested based on previous results)

  • Assess changes in ANO4 localization, clustering, or expression

  • Quantify changes using appropriate image analysis methods

• Phosphorylation-State Specific Analysis:

  • Combine ANO4 antibodies with phospho-specific antibodies

  • Investigate how calcium signaling affects ANO4 phosphorylation state

  • Use phosphatase inhibitors to preserve transient modifications

These approaches can provide valuable insights into the functional interplay between calcium signaling and ANO4 activity in various cell types and physiological conditions.

What role does ANO4 antibody play in understanding the involvement of ANO4 in disease models and potential therapeutic development?

ANO4 antibodies serve as critical tools for investigating the role of ANO4 in disease pathophysiology and therapeutic development:

Disease Biomarker Evaluation:

• Tissue Microarray Analysis:

  • Apply ANO4 antibody to tissue microarrays containing multiple patient samples

  • Correlate ANO4 expression with disease progression and patient outcomes

  • Assess potential as a diagnostic or prognostic biomarker

  • Recommended dilution for IHC applications: 1:20-1:200

• Liquid Biopsy Development:

  • Use ANO4 antibodies in immunoassays to detect shed/circulating ANO4

  • Evaluate correlation with disease state or treatment response

  • Compare with other established biomarkers

Therapeutic Target Validation:

• Target Engagement Studies:

  • Apply ANO4 antibody to verify binding of therapeutic candidates to ANO4

  • Use competition assays to map binding sites of potential drugs

  • Confirm specificity using appropriate controls

• Mechanism of Action Investigations:

  • Use ANO4 antibodies to track changes in expression, localization, or post-translational modifications in response to therapeutic interventions

  • Apply in both in vitro and in vivo disease models

  • Correlate molecular changes with functional outcomes

Therapeutic Development Applications:

• Antibody-Based Therapeutics:

  • Use experience with research-grade antibodies to inform development of therapeutic antibodies targeting ANO4

  • Test effects of antibody binding on ANO4 function

  • Assess potential for antibody-drug conjugates if ANO4 shows internalization

• High-Throughput Screening Support:

  • Develop cell-based assays with ANO4 antibody readouts

  • Screen compound libraries for modulators of ANO4 expression or localization

  • Validate hits with functional assays

For example, research utilizing ANO4 antibodies has already contributed to understanding ANO4's potential role in conditions such as epilepsy and cardiac arrhythmias, where chloride channel dysfunction is implicated in disease pathophysiology .

How can ANO4 antibodies contribute to understanding the evolutionary conservation and structural features of the Anoctamin family?

ANO4 antibodies provide valuable tools for comparative studies of Anoctamin family members across species and structural investigations:

Evolutionary Conservation Analysis:

• Cross-Species Reactivity Testing:

  • Test ANO4 antibody reactivity across species (human, mouse, rat, non-human primates)

  • Identify conserved epitopes through sequence alignment and antibody binding studies

  • Correlate epitope conservation with functional domain conservation

  • Use this information to infer evolutionary pressure on different protein regions

• Comparative Expression Profiling:

  • Apply ANO4 antibody to tissue panels from different species

  • Compare expression patterns to identify conserved vs. divergent expression

  • Correlate with functional studies to understand evolutionary adaptations

Structural Investigation Support:

• Domain-Specific Antibody Mapping:

  • Compare antibodies targeting different ANO4 domains

  • PACO35890 targets the N-terminal region (amino acids 1-200)

  • Use domain-specific antibodies to probe accessibility of different regions

  • Correlate with structural predictions and models

• Conformational Studies:

  • Develop conformation-specific antibodies that recognize active vs. inactive states

  • Use these tools to track conformational changes in response to calcium or other stimuli

  • Apply to understand structure-function relationships

Family-Wide Comparative Studies:

• Specificity Profiling:

  • Test cross-reactivity of ANO4 antibody against other Anoctamin family members

  • Use information to identify unique vs. shared epitopes

  • Develop specific immunoassays for distinguishing between family members

• Co-Expression Analysis:

  • Combine ANO4 antibody with antibodies against other Anoctamin family members

  • Investigate co-expression patterns in different tissues

  • Assess potential for heteromeric complex formation

These applications help build a comprehensive understanding of the evolutionary relationships, structural features, and functional specializations within the Anoctamin family, providing context for ANO4-specific findings.

What are common pitfalls in ANO4 antibody applications and how can researchers address them?

Researchers working with ANO4 antibodies may encounter several challenges, each requiring specific troubleshooting approaches:

Western Blot Challenges:

Immunostaining Challenges:

General Troubleshooting Approaches:

• Antibody Validation Controls:

  • Include positive control samples known to express ANO4

  • Use negative controls with ANO4 knockdown/knockout

  • Perform peptide competition assays to verify specificity

• Sample Preparation Considerations:

  • For membrane proteins like ANO4, avoid harsh detergents that may destroy epitopes

  • Consider native vs. denaturing conditions based on epitope accessibility

  • Use fresh samples when possible to minimize protein degradation

• Technical Optimization:

  • Systematically test different protocol parameters

  • Document all experimental conditions thoroughly

  • Maintain consistency across experimental replicates

By implementing these troubleshooting strategies, researchers can overcome common challenges and generate reliable data with ANO4 antibodies.

How should researchers interpret conflicting data from different ANO4 antibody applications?

When faced with discrepancies between different ANO4 antibody applications or results, researchers should follow a structured approach to data interpretation:

Sources of Discrepancies and Resolution Strategies:

• Epitope Accessibility Differences:

  • Different applications expose different epitopes (native vs. denatured)

  • Solution: Use multiple antibodies targeting distinct epitopes

  • Compare results from antibodies recognizing different domains (N-terminal vs. C-terminal)

  • The PACO35890 antibody targets amino acids 1-200, which may be differently accessible in various applications

• Technical vs. Biological Variability:

  • Distinguish between technical artifacts and true biological differences

  • Solution: Increase biological and technical replicates

  • Quantify variability across replicates

  • Apply appropriate statistical tests to determine significance

• Antibody Performance Differences:

  • Different antibodies may have varying specificity and sensitivity profiles

  • Solution: Compare antibody performance with validation controls

  • Consider using orthogonal techniques (e.g., RNA analysis, mass spectrometry)

  • Document antibody lot numbers and standardize protocols

Systematic Reconciliation Approach:

• Hierarchical Validation:

  • Establish a hierarchy of reliability based on validation evidence

  • Prioritize results from applications with stronger validation data

  • Use genetic controls (knockouts, knockdowns) as ultimate arbiters of specificity

• Correlation Analysis:

  • Quantitatively correlate results from different methods where possible

  • Look for patterns consistent across subset of techniques

  • Identify outlier techniques and investigate methodological explanations

• Contextual Interpretation:

  • Consider biological context when interpreting discrepancies

  • Different cell types may process ANO4 differently (expression level, PTMs, localization)

  • Developmental timing may affect epitope accessibility or expression patterns

By systematically analyzing and reconciling conflicting data, researchers can develop a more comprehensive and accurate understanding of ANO4 biology while acknowledging the technical limitations of antibody-based approaches.

What statistical approaches are recommended for analyzing ANO4 expression data across different experimental conditions?

Quantitative Data Analysis Framework:

• Preprocessing and Normalization:

  • For Western blot: Normalize ANO4 signal to appropriate loading controls

  • For IHC/IF: Apply background subtraction and normalization to reference markers

  • For high-throughput data: Implement batch correction methods

  • Always check data distribution before selecting statistical tests

• Appropriate Statistical Tests:

  • For comparing two conditions: Student's t-test (parametric) or Mann-Whitney test (non-parametric)

  • For multiple comparisons: ANOVA with appropriate post-hoc tests (Tukey, Bonferroni, or Dunnett)

  • For correlation analysis: Pearson's (linear) or Spearman's (non-parametric) correlation

  • For time-course studies: Repeated measures ANOVA or mixed-effects models

Advanced Analytical Approaches:

• Multivariate Analysis:

  • Principal Component Analysis (PCA) for identifying major sources of variation

  • Hierarchical clustering to identify expression patterns across conditions

  • Machine learning approaches for complex pattern recognition

• Spatial Statistics for Tissue Analysis:

  • Nearest neighbor analysis for clustering patterns

  • Ripley's K-function for spatial distribution analysis

  • Moran's I for spatial autocorrelation

Reporting Standards:

Special Considerations for ANO4 Studies:

• Account for tissue and cell-type heterogeneity in expression studies

• Consider non-normal distributions common in membrane protein expression data

• Implement appropriate controls for antibody lot-to-lot variability

• For longitudinal studies, account for repeated measures and time-dependent effects

By applying these rigorous statistical approaches, researchers can enhance the reliability and reproducibility of their ANO4 expression studies across different experimental conditions.