IVNS1ABP Antibody, HRP conjugated

Basic Research Questions

• What is IVNS1ABP and what is its functional significance in virus-host interactions?

IVNS1ABP (Influenza Virus NS1A Binding Protein) is a 642 amino acid protein that localizes to both the nucleus and cytoplasm. It contains one BACK domain, one BTB (POZ) domain, and six Kelch repeats. IVNS1ABP functions as a homodimer connected via its BTB domain and associates with F-Actin, playing a crucial role in organizing and stabilizing the actin cytoskeleton .

The protein was initially identified through its interaction with the influenza A virus nonstructural NS1 protein, which is relocalized in the nuclei of infected cells . Beyond viral interactions, IVNS1ABP participates in multiple cellular processes including regulation of cell division, pre-mRNA splicing, activation of the ERK signaling pathway, and protection of neuronal dendritic spines .

For research applications, understanding these functions is essential when investigating virus-host interactions or cytoskeletal dynamics in various experimental systems.

• What are the optimal applications for HRP-conjugated IVNS1ABP antibodies compared to unconjugated versions?

HRP-conjugated IVNS1ABP antibodies offer several advantages for specific detection applications:

• Direct detection: HRP conjugation eliminates the need for secondary antibody incubation, reducing protocol time and potential cross-reactivity issues .

• Enhanced sensitivity: The enzymatic amplification of signal from HRP provides excellent detection sensitivity, particularly valuable for proteins expressed at low levels.

• Optimal applications: HRP-conjugated IVNS1ABP antibodies are particularly suitable for ELISA and Western blotting applications, with demonstrated efficacy in IHC(P) when used at appropriate dilutions .

When comparing to unconjugated antibodies:

For multicolor applications or when signal amplification through secondary systems is desired, unconjugated antibodies may be preferable despite the additional protocol steps.

• What is the recommended protocol for antigen retrieval when using anti-IVNS1ABP antibodies in immunohistochemistry?

Based on validated protocols, optimal antigen retrieval for IVNS1ABP immunodetection in tissue sections follows these methodological steps:

• Buffer selection: Heat-mediated antigen retrieval in EDTA buffer (pH 8.0) has been extensively validated across multiple tissue types including ovarian cancer, glioma, placenta, lymphoma, thyroid cancer, and colon tissues .

• Blocking conditions: Block tissue sections with 10% goat serum to minimize non-specific binding. This concentration has been optimized for IVNS1ABP detection .

• Primary antibody incubation:

  • For IHC: Incubate sections with 2 μg/mL anti-IVNS1ABP antibody overnight at 4°C .

  • For IF: Use slightly higher concentration (5 μg/mL) when performing immunofluorescence detection .

• Secondary detection system:

  • For HRP-conjugated primary antibodies: Direct visualization with DAB substrate.

  • For unconjugated primaries: Use peroxidase-conjugated goat anti-rabbit IgG (for rabbit host antibodies) with 30-minute incubation at 37°C .

This protocol has been validated across diverse tissue types, demonstrating consistent and specific IVNS1ABP detection.

• What are the storage and handling recommendations to maintain optimal activity of IVNS1ABP antibodies?

Proper storage and handling of IVNS1ABP antibodies is critical for maintaining their specificity and activity over time:

• Reconstitution: For lyophilized antibodies, reconstitute in PBS buffer with 2% sucrose. Add 100 μL of distilled water to achieve a final antibody concentration of 1 mg/mL .

• Storage temperature: Store at -20°C or below for long-term stability . HRP-conjugated antibodies may have special storage requirements to preserve enzymatic activity.

• Aliquoting: Divide into small, single-use aliquots immediately after reconstitution to avoid repeated freeze-thaw cycles, which can significantly degrade antibody performance .

• Working dilutions: Prepare fresh working dilutions on the day of the experiment. IVNS1ABP antibodies typically perform optimally for:

  • ELISA at 1:312,500 dilution

  • Western blot at 2.0 μg/mL

• Avoid contamination: Work with sterile pipette tips and containers to prevent microbial contamination that could degrade antibody performance and introduce experimental artifacts.

Following these handling practices will help ensure consistent experimental results and maximize the usable lifespan of your antibody reagents.

Advanced Research Questions

• How can researchers optimize Western blot protocols specifically for IVNS1ABP detection in different cellular compartments?

IVNS1ABP localizes to both nuclear and cytoplasmic compartments, requiring optimization of Western blot protocols for comprehensive detection:

• Subcellular fractionation approach:

  • For complete detection, perform both whole cell lysate analysis and nuclear/cytoplasmic fractionation.

  • Validated positive controls include HeLa nuclear extract, MOLT-4 nuclear extract, and 3611-RF whole cell lysate .

• Sample preparation optimization:

  • For nuclear fraction: Use specialized nuclear extraction buffers containing DNase to reduce viscosity.

  • For cytoskeletal-associated fraction: Include cytoskeleton stabilizing buffers with protease inhibitors to preserve IVNS1ABP-actin interactions.

• Gel percentage selection:

  • Use 8-10% SDS-PAGE gels for optimal resolution of the 70 kDa IVNS1ABP protein .

  • Consider gradient gels (4-15%) when analyzing potential binding partners simultaneously.

• Transfer and detection parameters:

  • For HRP-conjugated anti-IVNS1ABP: Direct detection at 2.0 μg/mL dilution.

  • For unconjugated antibodies: Use m-IgGk BP-HRP secondary antibody at 1:1000-1:10000 dilution range .

  • Extended transfer times (1 hour at 100V or overnight at 30V) improve transfer efficiency of the 70 kDa protein.

• Validation approach:

  • Always include parallel samples with siRNA knockdown controls (sc-75349 for human, sc-75350 for mouse) to confirm specificity.

  • Consider using both monoclonal and polyclonal antibodies targeting different epitopes to validate detection.

These optimizations enable reliable detection of IVNS1ABP across different cellular compartments while minimizing artifacts.

• What are the methodological considerations for using IVNS1ABP antibodies in co-localization studies with cytoskeletal markers?

Given IVNS1ABP's association with F-actin and its role in cytoskeletal organization, co-localization studies require careful methodological planning:

• Fixation protocol selection:

  • Paraformaldehyde fixation (4%) preserves most cytoskeletal structures while maintaining IVNS1ABP epitope accessibility.

  • Avoid methanol fixation which can disrupt F-actin structures and potentially alter IVNS1ABP localization patterns.

• Antibody compatibility assessment:

  • When combining with phalloidin staining for F-actin, use IVNS1ABP antibodies conjugated to fluorophores with minimal spectral overlap (e.g., FITC-conjugated IVNS1ABP antibody with rhodamine-phalloidin).

  • For triple labeling, consider using IVNS1ABP antibodies available in multiple conjugates such as Alexa Fluor® 488, Alexa Fluor® 546, or phycoerythrin .

• Image acquisition parameters:

  • Collect z-stack images at Nyquist sampling rate to enable deconvolution and accurate co-localization analysis.

  • Use sequential scanning rather than simultaneous acquisition to minimize bleed-through artifacts.

• Quantitative co-localization analysis:

  • Apply both Pearson's correlation coefficient and Manders' overlap coefficient analyses.

  • Implement threshold controls using IVNS1ABP siRNA knockdown samples (sc-75349 for human, sc-75350 for mouse) .

• Validation experiments:

  • Include cytoskeleton disrupting agents (latrunculin, cytochalasin D) as controls to verify functional association between IVNS1ABP and F-actin.

  • Consider super-resolution microscopy (STED, STORM) for nanoscale co-localization assessment.

This methodological approach enables robust characterization of IVNS1ABP interactions with cytoskeletal components at both qualitative and quantitative levels.

• How does epitope specificity differ among available IVNS1ABP antibodies and how might this affect experimental outcomes?

Different commercial IVNS1ABP antibodies target distinct epitopes, which significantly impacts their utility in various experimental applications:

Key methodological considerations:

• Domain-specific detection:

  • BTB domain interactions: Best detected with antibodies targeting AA 1-300.

  • BACK domain and Kelch repeat functions: Better assessed with antibodies targeting AA 401-642.

• Post-translational modification impact:

  • Phosphorylation or ubiquitination may mask certain epitopes.

  • C-terminal antibodies may provide more consistent detection regardless of N-terminal modifications.

• Experimental validation approach:

  • When studying novel IVNS1ABP functions, validate with antibodies targeting different regions.

  • For interaction studies, select antibodies whose epitopes do not overlap with binding partner interaction sites.

• Isoform considerations:

  • N-terminal antibodies may detect all isoforms while C-terminal antibodies may be more specific to full-length IVNS1ABP.

Understanding these differences is crucial for experimental design, particularly when investigating protein-protein interactions or post-translational modifications affecting specific domains.

• What are the most effective strategies for validating IVNS1ABP antibody specificity in experimental settings?

Rigorous validation of IVNS1ABP antibody specificity is critical for generating reliable research data. The following comprehensive validation strategy is recommended:

• Genetic knockdown/knockout controls:

  • Implement IVNS1ABP siRNA (human: sc-75349, mouse: sc-75350)

  • Use shRNA plasmids (human: sc-75349-SH, mouse: sc-75350-SH)

  • Include CRISPR/Cas9 knockout cell lines when available

• Peptide competition assays:

  • Pre-incubate antibody with immunizing peptide at increasing concentrations

  • Demonstrate dose-dependent loss of signal specificity

  • Include unrelated peptide controls to confirm specificity

• Cross-platform validation methodology:

  • Compare results across multiple detection techniques (WB, IHC, IF)

  • Verify consistent molecular weight (70 kDa) across different sample types

  • Compare detection patterns between monoclonal and polyclonal antibodies targeting different epitopes

• Cross-species reactivity assessment:

  • Test against samples from multiple species (human, mouse, dog, etc.)

  • Document species-specific banding patterns or staining distributions

  • Verify sequence homology in the antibody epitope region across tested species

• Quantitative validation metrics:

  • Calculate signal-to-noise ratios in various applications

  • Perform titration experiments to determine optimal concentration ranges

  • Generate standard curves using recombinant protein standards

Implementation of this multi-faceted validation approach significantly enhances confidence in experimental findings and facilitates troubleshooting when unexpected results occur.

• How can IVNS1ABP antibodies be optimally employed to study host-pathogen interactions during influenza infection?

Given IVNS1ABP's role in influenza virus NS1 protein interactions, specialized methodological approaches are needed to study this host-pathogen relationship:

• Infection time-course analysis protocol:

  • Prepare influenza virus-infected and mock-infected control samples

  • Collect samples at multiple timepoints post-infection (4h, 8h, 12h, 24h)

  • Process parallel samples for both immunofluorescence microscopy and biochemical fractionation

• Subcellular relocalization assessment:

  • Perform nuclear/cytoplasmic fractionation of infected cells

  • Apply Western blot analysis using HRP-conjugated IVNS1ABP antibody (2.0 μg/mL)

  • Complement with immunofluorescence using 5 μg/mL antibody concentration

  • Quantify nuclear/cytoplasmic signal ratio changes over infection time-course

• Co-immunoprecipitation optimization:

  • Use agarose-conjugated IVNS1ABP antibodies for pull-down experiments

  • Include RNase treatment controls to distinguish RNA-dependent interactions

  • Analyze precipitates for NS1 protein and potential complex components

  • Compare results between different influenza virus strains

• Proximity ligation assay approach:

  • Combine anti-IVNS1ABP with anti-NS1 antibodies in dual-recognition format

  • Visualize direct interactions through fluorescent signal amplification

  • Quantify interaction frequency in different cellular compartments

• Mutational analysis methodology:

  • Express wild-type and domain-mutant versions of IVNS1ABP

  • Compare binding efficiencies to NS1 protein using immunoprecipitation

  • Correlate binding efficiency with viral replication outcomes

This comprehensive approach enables detailed characterization of the dynamic interactions between IVNS1ABP and viral components throughout the infection cycle.

• What methodological considerations are important when using IVNS1ABP antibodies for analyzing protein-protein interaction networks?

IVNS1ABP functions within complex protein interaction networks involving both cytoskeletal components and regulatory factors. The following methodological framework optimizes investigation of these networks:

• Crosslinking immunoprecipitation (CLIP) approach:

  • Apply UV crosslinking to stabilize transient interactions

  • Use agarose-conjugated IVNS1ABP antibodies for efficient complex isolation

  • Include stringent wash steps with varying salt concentrations to distinguish primary and secondary interactions

  • Analyze by mass spectrometry for unbiased interaction mapping

• Proximity-dependent biotinylation methodology:

  • Generate IVNS1ABP-BioID or IVNS1ABP-TurboID fusion constructs

  • Express in relevant cell types with appropriate controls

  • Capture biotinylated proximal proteins and analyze by Western blot using HRP-conjugated IVNS1ABP antibody (2.0 μg/mL)

  • Compare interactome under normal vs. stress conditions

• Domain-specific interaction mapping:

  • Select antibodies targeting specific IVNS1ABP domains (BTB domain, BACK domain, Kelch repeats)

  • Compare immunoprecipitation profiles to identify domain-specific binding partners

  • Validate key interactions with reciprocal co-immunoprecipitation experiments

• Quantitative interaction analysis:

  • Apply FRET or BRET approaches with fluorescently-tagged IVNS1ABP

  • Measure interaction dynamics in live cells under various conditions

  • Correlate with fixed-cell antibody-based detection methods

• Functional validation strategy:

  • Implement siRNA knockdown of IVNS1ABP and putative interaction partners

  • Assess effects on cytoskeletal organization, ERK signaling, and pre-mRNA splicing

  • Use rescue experiments with domain-specific mutants to map functional interactions

This comprehensive methodology enables robust characterization of IVNS1ABP interaction networks while minimizing artifacts and misinterpretations.