ASTN2 Antibody, HRP conjugated

What is ASTN2 and what is its significance in neurobiology?

ASTN2 (Astrotactin 2) is a protein primarily expressed in the brain that plays crucial roles in neuronal development and function. Research indicates that ASTN2 mediates recycling of the neuronal cell adhesion molecule ASTN1 to the anterior pole of the cell membrane in migrating neurons . It promotes ASTN1 internalization and intracellular transport of endocytosed ASTN1 . Additionally, ASTN2 selectively binds to specific phosphoinositides, including inositol-4,5-bisphosphate, inositol-3,4,5-trisphosphate, and inositol-1,3,4,5-tetrakisphosphate, suggesting its recruitment to membranes containing these lipid headgroups . Notably, deletions at the ASTN2 locus have been associated with schizophrenia, highlighting its potential role in neuropsychiatric disorders . Recent studies have also demonstrated that ASTN2 interacts with members of the Neuroligin family and modulates synaptic strength through trafficking and degradation of synaptic proteins .

What are the key specifications of commercially available ASTN2 Antibody (HRP conjugated)?

Commercially available ASTN2 Antibody (HRP conjugated) typically presents as a rabbit polyclonal antibody with reactivity against human ASTN2 . The antibody is directly conjugated to horseradish peroxidase (HRP), eliminating the need for secondary antibodies in detection protocols . Key specifications include:

Some products are generated using specific immunogens, such as recombinant human Astrotactin-2 protein (amino acids 108-272) , which should be considered when selecting the appropriate antibody for targeting specific domains of ASTN2.

How does the HRP conjugation function in antibody detection systems?

The HRP conjugation provides a direct detection system through enzymatic amplification of signal. Horseradish peroxidase catalyzes the oxidation of substrates in the presence of hydrogen peroxide, resulting in either colored precipitates (for chromogenic detection) or light emission (for chemiluminescent detection) . This enzymatic reaction significantly enhances detection sensitivity compared to unconjugated primary antibodies.

In experimental protocols, the HRP-conjugated ASTN2 antibody binds directly to the target protein, and upon addition of an appropriate substrate, the HRP enzyme generates a detectable signal . For Western blotting applications, chemiluminescent substrates like ECL (Enhanced Chemiluminescence) reagents produce light that can be captured on film or with digital imaging systems . For ELISA and immunohistochemistry, chromogenic substrates like TMB (3,3',5,5'-tetramethylbenzidine) or DAB (3,3'-diaminobenzidine) produce colored precipitates visible to the naked eye or through microscopy .

The primary advantages of using HRP-conjugated antibodies include increased sensitivity through signal amplification, reduced protocol time by eliminating secondary antibody incubation steps, and decreased background from non-specific secondary antibody binding .

How should researchers optimize ASTN2 Antibody (HRP conjugated) for Western blot applications?

Optimizing ASTN2 Antibody (HRP conjugated) for Western blot requires systematic adjustment of several parameters:

• Antibody Dilution: Begin with a titration series, typically ranging from 1:2000 to 1:10000 . Test multiple dilutions simultaneously to determine the optimal concentration that provides robust specific signal with minimal background.

• Protein Loading: For brain tissue or neuronal cultures where ASTN2 is expressed, load 25μg of total protein per lane as a starting point . Adjust based on expression levels in your specific samples.

• Blocking Conditions: Use 3% non-fat dry milk in TBST as an initial blocking buffer . If background issues persist, alternative blocking agents such as BSA or commercial blocking solutions can be tested.

• Incubation Parameters: Incubate membranes with diluted antibody overnight at 4°C with gentle agitation for optimal binding. For quick protocols, room temperature incubation for 1-2 hours may be sufficient but might reduce sensitivity.

• Detection System: Select an appropriate chemiluminescent substrate based on expected signal strength. ECL Basic Kit provides standard detection sensitivity , while enhanced substrates offer greater sensitivity for detecting low abundance targets.

• Exposure Time: Begin with short exposures (5-10 seconds) and incrementally increase until optimal signal-to-noise ratio is achieved. Avoid overexposure which can mask specific bands.

• Expected Molecular Weight: When analyzing results, look for the ASTN2 protein at its predicted molecular weight, considering potential post-translational modifications that may alter migration patterns.

For validation, include positive controls (tissues known to express ASTN2, such as brain lysates) and negative controls (tissues or cell lines with minimal ASTN2 expression) .

What protocols are recommended for ELISA applications using ASTN2 Antibody (HRP conjugated)?

For ELISA applications with ASTN2 Antibody (HRP conjugated), the following protocol framework is recommended:

• Plate Coating:

  • For direct ELISA: Coat 96-well plates with target antigen (purified ASTN2 protein or sample containing ASTN2) at 1-10 μg/mL in carbonate/bicarbonate buffer (pH 9.6).

  • For sandwich ELISA: Coat with a capture antibody targeting a different ASTN2 epitope than the HRP-conjugated detection antibody.

  • Incubate overnight at 4°C.

• Blocking:

  • Block remaining protein-binding sites with 1-5% BSA or non-fat dry milk in PBS.

  • Incubate for 1-2 hours at room temperature.

• Sample Addition:

  • Add diluted samples and standards to appropriate wells.

  • Incubate for 1-2 hours at room temperature or overnight at 4°C.

• Antibody Addition:

  • Add ASTN2 Antibody (HRP conjugated) diluted to approximately 1 μg/mL in blocking buffer .

  • Titrate to determine optimal concentration for your specific experimental conditions.

  • Incubate for 1-2 hours at room temperature.

• Detection:

  • Add appropriate HRP substrate (TMB for colorimetric detection).

  • Monitor color development (typically 5-30 minutes).

  • Stop reaction with 2N H₂SO₄ or other suitable stop solution.

  • Read absorbance at appropriate wavelength (450 nm for TMB).

• Critical Parameters:

  • Include thorough washing steps between each stage (5-6 washes with PBS-T).

  • Run appropriate controls including blank wells, negative controls, and standard curves using recombinant ASTN2 protein.

  • For quantitative analysis, generate standard curves using purified ASTN2 protein at known concentrations.

This protocol provides a foundational framework that should be optimized for specific experimental requirements, sample types, and detection sensitivity needs .

What are the methodological considerations for investigating ASTN2's interactions with Neuroligins?

Investigating ASTN2's interactions with Neuroligins using HRP-conjugated antibodies requires specialized methodologies that leverage both the specificity of the antibody and the sensitivity of HRP detection. Research has shown that ASTN2 interacts with multiple Neuroligin family members with varying affinities, with full-length ASTN2 binding more strongly to NLGN1/2 and truncated versions showing stronger interactions with NLGN3/4 . The following methodological approaches are recommended:

• Co-immunoprecipitation Analysis:

  • Immunoprecipitate ASTN2 from neuronal lysates or heterologous expression systems.

  • Probe Western blots with antibodies against different Neuroligin family members.

  • Alternatively, immunoprecipitate Neuroligins and probe with ASTN2 Antibody (HRP conjugated).

  • Use stringent washing conditions to confirm specificity of interactions .

• Surface Expression Analysis:

  • Utilize live immunolabeling and flow cytometry to quantify surface expression of tagged Neuroligins in the presence and absence of ASTN2 overexpression .

  • Compare rates of internalization between control conditions and ASTN2 overexpression conditions using pulse-chase experiments.

• Trafficking Analysis:

  • Employ time-resolved imaging to track the co-localization of ASTN2 and Neuroligins during endocytosis.

  • Utilize endosomal markers to determine compartment localization following internalization.

  • Correlate ASTN2 expression levels with Neuroligin degradation rates using cycloheximide chase experiments.

• Domain Mapping:

  • Use truncation or point mutation variants of ASTN2, particularly targeting the FNIII domain, to determine regions critical for Neuroligin binding.

  • Compare binding profiles of full-length ASTN2 versus JDUP (a truncated version lacking the FNIII domain) with different Neuroligin family members .

• Functional Analysis:

  • Assess electrophysiological consequences of altered ASTN2-Neuroligin interactions through patch-clamp recordings.

  • Compare NLGN2 expression levels in neuronal soma following ASTN2-EGFP or JDUP-EGFP overexpression .

These methodological approaches provide complementary data on both the physical interactions and functional consequences of ASTN2-Neuroligin binding, offering insights into how these proteins cooperate in neuronal development and synaptic function.

What are common issues and solutions when using ASTN2 Antibody (HRP conjugated) in Western blotting?

Researchers may encounter several issues when using ASTN2 Antibody (HRP conjugated) in Western blotting. Here are common problems and their solutions:

• Weak or No Signal:

  • Possible Causes: Insufficient antibody concentration, inadequate antigen amount, inefficient transfer, compromised antibody activity, or low target expression.

  • Solutions:

    • Increase antibody concentration (decrease dilution factor)

    • Load more protein (30-50 μg)

    • Verify transfer efficiency with reversible protein stains

    • Check antibody storage conditions (avoid repeated freeze-thaw cycles)

    • Use fresh detection substrate

    • Extend exposure time during imaging

    • Include positive controls from tissues known to express ASTN2 (brain tissue)

• High Background:

  • Possible Causes: Excessive antibody concentration, insufficient blocking, inadequate washing, or cross-reactivity.

  • Solutions:

    • Increase antibody dilution (1:5000-1:10000)

    • Extend blocking time or try alternative blocking agents (BSA, commercial blockers)

    • Increase wash duration and frequency (5-6 washes, 5-10 minutes each)

    • Add 0.05-0.1% SDS to wash buffer to reduce non-specific binding

    • Reduce substrate development time

• Multiple Bands:

  • Possible Causes: Cross-reactivity, protein degradation, splice variants, or post-translational modifications.

  • Solutions:

    • Use fresh samples with complete protease inhibitors

    • Verify band patterns with different antibodies targeting distinct ASTN2 epitopes

    • Research known ASTN2 splice variants or modifications in your experimental system

    • Perform antibody validation with ASTN2 knockdown or knockout controls

• Inconsistent Results:

  • Possible Causes: Variable sample preparation, antibody degradation, or inconsistent transfer.

  • Solutions:

    • Standardize sample collection and processing

    • Aliquot antibody to avoid repeated freeze-thaw cycles

    • Use internal loading controls

    • Maintain consistent transfer conditions

    • Document and standardize exposure settings

• Unexpected Molecular Weight:

  • Possible Causes: Post-translational modifications, splice variants, or sample preparation issues.

  • Solutions:

    • Research known ASTN2 molecular weight variations

    • Try different sample preparation methods (different lysis buffers)

    • Compare with recombinant ASTN2 protein standards

    • Verify with mass spectrometry if available

Methodical troubleshooting by systematically adjusting these parameters should resolve most issues encountered with ASTN2 Antibody (HRP conjugated) in Western blotting applications.

How should researchers optimize storage and handling of ASTN2 Antibody (HRP conjugated) to maintain activity?

Proper storage and handling of ASTN2 Antibody (HRP conjugated) is critical for maintaining its activity and ensuring consistent experimental results. The following practices are recommended:

• Storage Temperature:

  • Store at -20°C in a non-frost-free freezer to prevent temperature fluctuations .

  • Never store at room temperature or 4°C for extended periods as this accelerates HRP degradation.

• Aliquoting:

  • Upon receipt, divide the antibody into small single-use aliquots to minimize freeze-thaw cycles .

  • Use sterile microcentrifuge tubes for aliquoting and label with antibody information and date.

• Buffer Composition:

  • The antibody is typically supplied in 0.01 M PBS, pH 7.4, with 0.03% Proclin-300 and 50% Glycerol .

  • Do not alter this buffer composition unless specifically required for your application.

• Protection from Light:

  • HRP is light-sensitive; keep the antibody protected from light during storage and handling .

  • Use amber tubes for storage or wrap tubes in aluminum foil.

• Avoid Freeze-Thaw Cycles:

  • Repeated freezing and thawing significantly reduces antibody activity .

  • Thaw aliquots only once and discard any unused portion.

• Handling During Experiments:

  • Thaw antibody on ice immediately before use.

  • Keep on ice while preparing dilutions.

  • Return to -20°C promptly after use.

  • Prepare working dilutions fresh for each experiment.

• Contamination Prevention:

  • Use clean pipette tips for each handling.

  • Avoid introducing bacteria or fungi which can degrade the antibody or affect HRP activity.

  • Never pipette directly from the stock tube; always transfer the needed amount to a separate tube first.

• Shelf-Life Monitoring:

  • Track antibody performance over time.

  • Consider re-validation after extended storage periods (>6 months).

  • Note any decrease in signal strength which may indicate activity loss.

• Shipping and Temporary Transport:

  • If temporary transport is necessary, use dry ice or freezer packs.

  • Minimize time at temperatures above -20°C.

Following these guidelines will help maintain ASTN2 Antibody (HRP conjugated) activity and ensure reliable, reproducible experimental results throughout the antibody's usable lifespan .

What control experiments should be included when using ASTN2 Antibody (HRP conjugated)?

Implementing appropriate controls is essential for validating results obtained with ASTN2 Antibody (HRP conjugated). The following control experiments should be included:

• Positive Controls:

  • Include samples known to express ASTN2, such as brain tissue lysates or cerebellar granule cells .

  • Recombinant ASTN2 protein can serve as a defined positive control for antibody validation.

  • Cell lines with confirmed ASTN2 expression provide consistent positive controls across experiments.

• Negative Controls:

  • Samples from tissues known to have minimal ASTN2 expression.

  • For genetic model systems, ASTN2 knockdown or knockout samples provide ideal negative controls.

  • Primary antibody omission controls to assess non-specific binding of detection reagents.

• Specificity Controls:

  • Peptide competition assays where the antibody is pre-incubated with excess immunizing peptide before application to samples.

  • Comparison with alternative ASTN2 antibodies targeting different epitopes to confirm band patterns.

  • Immunoprecipitation followed by mass spectrometry to verify antibody specificity.

• Dilution Series:

  • Test multiple antibody dilutions (e.g., 1:2000, 1:5000, 1:10000) in parallel to determine optimal concentration .

  • Serial dilutions of sample to confirm signal proportionality to protein concentration.

• Loading Controls:

  • Include housekeeping protein detection (β-actin, GAPDH, α-tubulin) to normalize for loading variations.

  • Total protein staining methods (Ponceau S, REVERT) provide alternative normalization approaches.

• Application-Specific Controls:

  • For Western blotting: Molecular weight markers to confirm target band size.

  • For ELISA: Standard curves using recombinant ASTN2 protein at known concentrations.

  • For immunohistochemistry: Serial section controls with primary antibody omission.

• Expression Manipulation Controls:

  • Overexpression systems to confirm antibody detection of increased ASTN2 levels.

  • siRNA or shRNA knockdown to demonstrate reduced signal with reduced target expression.

  • CRISPR/Cas9-mediated knockout for complete elimination of specific signal.

• Cross-Reactivity Assessment:

  • Testing the antibody on related proteins (like ASTN1) to evaluate potential cross-reactivity.

  • Using samples from different species if working with non-human models to confirm reactivity.

These control experiments provide crucial validation of antibody performance and significantly enhance the reliability and interpretability of experimental findings when using ASTN2 Antibody (HRP conjugated) .

How can ASTN2 Antibody (HRP conjugated) be used to investigate neuronal migration mechanisms?

ASTN2 Antibody (HRP conjugated) provides valuable tools for investigating neuronal migration mechanisms through several advanced methodological approaches:

• Spatiotemporal Expression Analysis:

  • Use immunohistochemistry with HRP-conjugated ASTN2 antibody to map expression patterns during critical developmental windows when neuronal migration is active.

  • Compare expression between anterior and posterior poles of migrating neurons to confirm ASTN2's polarized distribution .

  • Conduct developmental time-course studies to correlate ASTN2 expression with migration phases.

• Co-localization with Migration Machinery:

  • Perform double-labeling experiments to assess co-localization of ASTN2 with components of the neuronal migration apparatus (cytoskeletal elements, motor proteins, cell adhesion molecules).

  • Specifically examine co-localization with ASTN1, as ASTN2 mediates recycling of ASTN1 to the anterior membrane of migrating neurons .

  • Investigate relationships with centrosomal proteins, which coordinate leading process extension during migration.

• Functional Migration Assays:

  • Combine antibody labeling with ex vivo or in vitro neuronal migration assays to correlate ASTN2 expression levels with migration rate and directionality.

  • Analyze ASTN2 distribution before and after application of migration-promoting or inhibiting factors.

  • Use cerebellum slice cultures where granule cell migration is well-characterized to study ASTN2 dynamics in a physiologically relevant system .

• Molecular Interaction Studies:

  • Examine interactions between ASTN2 and phosphoinositides, as ASTN2 selectively binds inositol-4,5-bisphosphate, inositol-3,4,5-trisphosphate, and inositol-1,3,4,5-tetrakisphosphate .

  • Investigate how these lipid interactions contribute to ASTN2's membrane recruitment during migration.

  • Assess how ASTN2's binding to these phosphoinositides relates to its function in ASTN1 recycling.

• Vesicular Trafficking Analysis:

  • Examine co-localization of ASTN2 with endosomal markers to understand its role in intracellular transport pathways during migration.

  • Investigate whether ASTN2 localizes to specific vesicular compartments during different phases of the migration cycle.

  • Analyze how disruption of various trafficking pathways affects ASTN2 distribution and function.

• Genetic Model Systems:

  • Compare ASTN2 distribution and neuronal migration patterns between wildtype and neurological disorder models (particularly schizophrenia models, given the association between ASTN2 deletions and this disorder) .

  • Utilize conditional knockout/knockdown systems to manipulate ASTN2 expression in specific neuronal populations during defined developmental windows.

These methodological approaches collectively provide insights into how ASTN2 contributes to neuronal migration mechanisms, particularly through its role in ASTN1 recycling and membrane protein trafficking .

What methodologies can investigate ASTN2's role in synaptic protein trafficking?

Investigating ASTN2's role in synaptic protein trafficking requires sophisticated methodological approaches that can track the movement and fate of synaptic proteins. The following methodologies utilizing ASTN2 Antibody (HRP conjugated) are recommended:

• Pulse-Chase Protein Trafficking Assays:

  • Express tagged synaptic proteins (e.g., Neuroligins) in neuronal cultures or HEK293 cells.

  • Track their degradation rates in the presence or absence of ASTN2 overexpression.

  • Use ASTN2 Antibody (HRP conjugated) to correlate ASTN2 expression with target protein turnover rates .

  • Quantify remaining protein levels at various time points after protein synthesis inhibition with cycloheximide.

• Subcellular Fractionation Analysis:

  • Separate neuronal lysates into membrane, cytosolic, synaptosomal, and vesicular fractions.

  • Use Western blotting with the HRP-conjugated antibody to track ASTN2's distribution across these fractions.

  • Correlate this distribution with the localization of interaction partners like Neuroligins .

  • Compare fractionation profiles between basal conditions and after synaptic activity stimulation.

• Surface Protein Internalization Assays:

  • Surface biotinylate neuronal proteins and allow internalization to occur over various timepoints.

  • Strip remaining surface biotin and isolate internalized biotinylated proteins.

  • Analyze how ASTN2 expression levels affect internalization rates of synaptic proteins.

  • Use ASTN2 Antibody (HRP conjugated) in Western blotting to correlate trafficking with ASTN2 levels .

• Co-localization with Endosomal Markers:

  • Perform immunocytochemistry using antibodies against endocytic pathway markers.

  • Include markers for early endosomes (EEA1), late endosomes (Rab7), recycling endosomes (Rab11), and lysosomes (LAMP1).

  • Convert HRP signal to fluorescent readout using tyramide signal amplification for multi-color imaging.

  • Quantify co-localization coefficients between ASTN2 and these markers in different cellular compartments.

• Proteasomal and Lysosomal Inhibition Studies:

  • Treat neurons with proteasome inhibitors (MG132) or lysosomal inhibitors (Bafilomycin A1).

  • Assess how inhibition affects levels of ASTN2-interacting partners like Neuroligins and ROCK2 .

  • Use ASTN2 Antibody (HRP conjugated) to determine if ASTN2 levels are also affected by these inhibitors.

  • This approach helps distinguish between proteasomal and lysosomal degradation pathways.

• Electrophysiological Correlates:

  • Perform electrophysiological recordings to measure synaptic strength following manipulation of ASTN2 levels.

  • Compare results between full-length ASTN2 and truncated versions like JDUP that lack the FNIII domain .

  • Correlate functional changes with ASTN2-mediated alterations in synaptic protein localization.

  • Specifically assess how ASTN2 overexpression affects miniature excitatory postsynaptic currents, which reflect synaptic strength.

These methodologies collectively provide a comprehensive analysis of how ASTN2 influences the trafficking and degradation of synaptic proteins, particularly adhesion molecules like Neuroligins, thereby modulating synaptic strength and neuronal communication .

How can researchers investigate ASTN2's role in neurological disorders using the HRP-conjugated antibody?

ASTN2 has been implicated in several neurological disorders, particularly schizophrenia where deletions at the ASTN2 locus have been identified . The HRP-conjugated ASTN2 antibody provides valuable tools for investigating these associations through several methodological approaches:

• Comparative Expression Analysis in Clinical Samples:

  • Quantify ASTN2 protein levels in post-mortem brain tissue from individuals with neurological disorders versus healthy controls using Western blot analysis.

  • The direct HRP conjugation enables sensitive detection even with limited sample quantities.

  • Compare expression patterns across brain regions implicated in specific disorders (e.g., prefrontal cortex in schizophrenia).

  • Correlate ASTN2 expression levels with other proteins implicated in the same disorders, such as ROCK2, which shows an inverse correlation with ASTN2 levels in some patient samples .

• Patient-Derived Cellular Models:

  • Generate induced pluripotent stem cell (iPSC)-derived neurons from patients with ASTN2-associated disorders.

  • Compare ASTN2 protein expression, localization, and degradation patterns with cells from healthy controls.

  • Assess whether disease-associated ASTN2 variants show altered trafficking of interaction partners like Neuroligins.

• Animal Models of Neurological Disorders:

  • Develop or utilize existing animal models carrying disease-associated ASTN2 variants or conditional knockouts.

  • Perform immunohistochemistry and Western blotting with the HRP-conjugated antibody to analyze how these genetic alterations affect ASTN2 expression, localization, and stability.

  • Correlate molecular findings with behavioral phenotypes relevant to human neurological conditions.

• Synaptic Dysfunction Analysis:

  • Examine how disease-associated ASTN2 variants affect synaptic protein levels and localization.

  • Particularly focus on Neuroligin family members, which interact with ASTN2 and are independently implicated in neurodevelopmental disorders.

  • Compare NLGN2 expression in neuronal soma between normal and disease conditions, as ASTN2 overexpression leads to decreased NLGN2 levels .

• Therapeutic Target Validation:

  • In experimental therapeutic approaches targeting ASTN2-related pathways, use the antibody to monitor changes in ASTN2 expression or localization as biomarkers of treatment efficacy.

  • Assess whether compounds that modulate ASTN2's interactions with Neuroligins might have therapeutic potential.

• Protein Interaction Network Analysis:

  • Perform co-immunoprecipitation followed by mass spectrometry to identify alterations in ASTN2's protein interaction network in disease states.

  • Compare these networks between healthy controls and disease models to identify dysregulated pathways.

  • Investigate whether these altered interaction patterns contribute to disease mechanisms.

• Genetic-Molecular Correlation Studies:

  • For patient populations with identified ASTN2 genetic variants, assess whether these variations correlate with altered protein expression patterns or interaction profiles.

  • This approach can help establish causative relationships between genetic variation and molecular dysfunction.

These methodological approaches collectively provide a comprehensive framework for investigating ASTN2's role in neurological disorders, potentially identifying novel therapeutic targets or diagnostic biomarkers .

What are the current limitations in ASTN2 antibody research and future directions?

Despite significant advances in understanding ASTN2's functions, several limitations exist in current ASTN2 antibody research that represent important areas for future development:

Current limitations include:

• Antibody Specificity Challenges: The significant sequence homology between ASTN1 and ASTN2 creates potential cross-reactivity issues that require rigorous validation controls.

• Isoform Specificity: Multiple transcript variants of ASTN2 have been identified , but current antibodies may not distinguish between these isoforms, limiting our understanding of their differential functions.

• Model System Constraints: Most ASTN2 research has focused on cerebellar systems , leaving its roles in other brain regions less characterized, despite evidence for broader expression patterns.

• Temporal Resolution Limitations: Current methodologies primarily provide static snapshots of ASTN2 localization and interactions, limiting our understanding of its dynamic trafficking behaviors.

• Mechanistic Gaps: While interactions with Neuroligins have been identified , the precise molecular mechanisms by which ASTN2 regulates their trafficking remain incompletely understood.

Future research directions should address these limitations through:

• Development of Isoform-Specific Antibodies: Generation of antibodies targeting unique epitopes of different ASTN2 isoforms would enable exploration of their specific functions.

• Advanced Imaging Approaches: Implementation of super-resolution microscopy and live-cell imaging with tagged ASTN2 would provide deeper insights into its dynamic behaviors during neuronal development and synaptic function.

• Expanded Disease Model Research: Investigation of ASTN2's roles in a broader range of neurological and psychiatric disorders beyond schizophrenia, particularly autism spectrum disorders where synaptic dysfunction is prominent.

• Structure-Function Analysis: Detailed mapping of ASTN2's functional domains and how they contribute to protein-protein interactions and trafficking activities.

• Therapeutic Targeting Strategies: Exploration of approaches to modulate ASTN2 function in disease contexts, potentially through small molecules that affect its protein-protein interactions or trafficking activities.

• Cross-Species Comparative Studies: Development of antibodies with broader species reactivity would facilitate evolutionary and comparative analyses of ASTN2 function across model organisms.

Addressing these limitations and pursuing these future directions will significantly enhance our understanding of ASTN2's roles in neuronal development, migration, and synaptic function, potentially leading to novel therapeutic approaches for associated neurological disorders.

How can researchers integrate multiple detection methods with ASTN2 Antibody (HRP conjugated) for comprehensive analysis?

Integrating multiple detection methods with ASTN2 Antibody (HRP conjugated) enables comprehensive analysis of this protein's expression, localization, interactions, and functions. The following integrated approach maximizes research outcomes:

• Multimodal Detection Strategy:

  • Combine Western blotting for quantitative expression analysis with immunohistochemistry/immunocytochemistry for spatial localization patterns.

  • Supplement with ELISA for high-throughput quantification across multiple samples or experimental conditions.

  • The HRP conjugation provides versatility across these methods through appropriate substrate selection .

• Sequential or Multiplexed Detection Protocols:

  • For tissue sections or cultured cells, implement tyramide signal amplification to convert HRP signal to a fluorescent readout.

  • This enables co-localization studies with other proteins using distinct fluorophores.

  • After HRP signal development, quench the peroxidase activity with hydrogen peroxide treatment and proceed with additional antibody labeling cycles .

• Correlative Microscopy Approaches:

  • Begin with light microscopy using HRP-based chromogenic detection to identify regions of interest.

  • Process the same samples for electron microscopy to analyze subcellular localization at ultrastructural resolution.

  • This approach bridges macro-scale expression patterns with nano-scale localization details.

• Functional-Molecular Correlation:

  • Combine electrophysiological recordings with post-hoc immunohistochemistry using ASTN2 Antibody (HRP conjugated).

  • This approach directly correlates functional properties with molecular expression patterns in the same cells .

  • Particularly valuable for understanding how ASTN2-mediated trafficking affects synaptic strength.

• Temporal Analysis Framework:

  • Implement experimental designs that capture both acute changes (minutes to hours) and long-term adaptations (days to weeks).

  • Use the same detection methods across different timepoints to ensure comparable results.

  • This approach reveals how ASTN2's roles may evolve throughout development or disease progression.

• Genetic-Biochemical Integration:

  • Combine genomic analyses (sequencing, SNP identification) with protein-level detection using the HRP-conjugated antibody.

  • This correlates genetic variants with potential alterations in protein expression, localization, or function.

  • Particularly valuable for understanding how ASTN2 genetic variants associated with neurological disorders manifest at the protein level.

• Multi-dimensional Data Analysis:

  • Implement advanced image analysis algorithms for quantitative assessment of ASTN2 distribution patterns.

  • Apply statistical approaches designed for multiple dependent variables to integrate data from diverse methodology platforms.

  • Utilize machine learning approaches to identify complex patterns across multidimensional datasets.