IVNS1ABP Antibody, FITC conjugated

What is IVNS1ABP and why is it significant in research?

IVNS1ABP is a 642 amino acid protein that contains one BACK domain, one BTB (POZ) domain, and six Kelch repeats. It functions as a homodimer connected via its BTB domain and plays critical roles in multiple cellular processes. Research significance stems from its involvement in:

• Pre-mRNA splicing and RNA processing

• Actin cytoskeleton organization and stabilization through F-actin association

• Aryl hydrocarbon receptor (AHR) pathway modulation

• Protein ubiquitination regulation

• Viral response mechanisms, particularly with influenza virus

The protein localizes to both nucleus and cytoplasm, making it relevant for studies of multiple cellular compartments and processes .

How does FITC conjugation enhance IVNS1ABP antibody applications?

FITC conjugation provides several methodological advantages in IVNS1ABP research:

• Direct visualization without secondary antibody requirements, reducing experimental complexity and potential cross-reactivity

• Excitation maximum at approximately 495 nm and emission maximum at 519 nm, compatible with standard filter sets in most fluorescence microscopes

• Enables multicolor immunofluorescence when combined with other fluorophores (e.g., PE, Alexa Fluor conjugates)

• Facilitates flow cytometry (FCM) analysis for quantitative assessment of IVNS1ABP expression across cell populations

For optimal FITC-conjugated antibody performance, samples should be protected from light during incubation and storage to prevent photobleaching.

What are the validated applications for IVNS1ABP antibody with FITC conjugation?

Based on manufacturer validation data and published research, FITC-conjugated IVNS1ABP antibodies can be reliably used for:

For each application, antigen retrieval methods and blocking protocols should be optimized based on tissue type and fixation method .

How should I optimize immunofluorescence protocols for IVNS1ABP localization studies?

For robust IVNS1ABP localization using FITC-conjugated antibodies:

• Fixation optimization: Compare 4% paraformaldehyde (10-15 minutes) versus methanol fixation (5 minutes at -20°C). Methanol fixation has been shown to better preserve cytoskeletal elements for IVNS1ABP visualization .

• Permeabilization: Use 0.1-0.3% Triton X-100 for 5-10 minutes for nuclear IVNS1ABP detection. For cytoplasmic focus, reduce permeabilization time to 3-5 minutes.

• Blocking strategy: Implement a dual blocking approach with 5% BSA and 5% normal serum (matching secondary antibody host if used in multiplex staining).

• Co-localization studies:

  • F-actin co-staining: Use rhodamine-phalloidin (1:200) to visualize IVNS1ABP association with actin filaments

  • Nuclear co-localization: DAPI counterstaining (1:1000) for 5 minutes to assess nuclear versus cytoplasmic distribution ratios

• Controls: Include unstained, secondary-only, and isotype controls alongside positive controls using cell lines with known IVNS1ABP expression (HeLa, MCF-7, U87-MG are validated positive controls) .

What considerations are important when designing Western blot experiments with IVNS1ABP antibodies?

For optimal Western blot detection of IVNS1ABP:

• Sample preparation:

  • Nuclear fractionation is recommended for enrichment as IVNS1ABP distributes between cytoplasmic and nuclear compartments

  • Use RIPA buffer with protease inhibitors, supplemented with 10mM N-ethylmaleimide to preserve ubiquitination status

• Electrophoresis parameters:

  • Expected molecular weight: ~70-72 kDa

  • 8-10% SDS-PAGE gels provide optimal resolution

  • Include positive control lysates: HeLa nuclear extract, MOLT-4 nuclear extract, or 3611-RF whole cell lysate

• Transfer and detection:

  • Semi-dry transfer: 15V for 60 minutes provides efficient transfer

  • Blocking: 5% non-fat milk in TBST for 1 hour at room temperature

  • Primary antibody dilution: 1:500-1:3000 determined through titration experiments

  • For fluorescent detection systems, secondary antibodies conjugated with compatible fluorophores (avoiding spectral overlap with FITC) should be considered

• Validation approach: The expected 70-72 kDa band should be validated through siRNA knockdown experiments to confirm specificity .

How can I optimize flow cytometry protocols for IVNS1ABP detection?

For flow cytometry applications using FITC-conjugated IVNS1ABP antibodies:

• Cell preparation considerations:

  • Fixation: 2% paraformaldehyde for 10-15 minutes at room temperature

  • Permeabilization: 0.1% saponin in PBS for intracellular staining

  • Cell concentration: 1×10^6 cells/mL is optimal for detection

• Staining protocol optimization:

  • Antibody dilution: Start at 1:100 and titrate to determine optimal signal-to-noise ratio

  • Incubation conditions: 30-45 minutes at room temperature in the dark

  • Washing buffer: PBS with 1% FBS and 0.1% sodium azide

• Instrument setup:

  • Use FITC channel (typically FL1 on many cytometers)

  • Compensation is critical when multiplexing with other fluorophores

  • Include single-color controls for each fluorophore

• Analytical approaches:

  • For expression level studies: Mean fluorescence intensity (MFI) comparison

  • For regulated expression: Fold-change in MFI under treatment conditions

  • For population analysis: Percent positive cells above isotype control threshold

How can IVNS1ABP antibodies be employed to study its role in influenza virus pathogenesis?

IVNS1ABP interacts with influenza virus NS1 protein, playing a critical role in viral replication. Advanced research approaches include:

• Co-immunoprecipitation studies:

  • Use FITC-conjugated IVNS1ABP antibodies to visualize co-localization with viral NS1 protein

  • Immunoprecipitate with anti-IVNS1ABP followed by Western blot detection of NS1

  • Validate interactions with proximity ligation assay (PLA) for in situ detection

• Functional analysis during infection:

  • Monitor IVNS1ABP relocalization during viral infection timepoints (0, 2, 4, 8, 12, 24 hours post-infection)

  • Conduct FRAP (Fluorescence Recovery After Photobleaching) experiments to assess dynamics

  • Implement IVNS1ABP knockdown/knockout to evaluate effects on viral replication

• Alternative splicing investigation:

  • IVNS1ABP facilitates alternative splicing of influenza A virus M1 mRNA through HNRNPK interaction

  • RT-PCR analysis of M1/M2 mRNA ratios following IVNS1ABP depletion

  • RNA-IP (RNA immunoprecipitation) to identify direct RNA targets

These approaches help delineate the mechanistic role of IVNS1ABP in viral replication and host-pathogen interactions.

What methodologies can be used to study IVNS1ABP's role in cytoskeletal dynamics?

For investigating IVNS1ABP's cytoskeletal functions:

• Live-cell imaging approaches:

  • Transfect cells with IVNS1ABP-GFP fusion constructs alongside LifeAct-RFP for real-time visualization

  • Implement photo-activatable IVNS1ABP constructs for spatiotemporal control

  • Use TIRF microscopy to visualize membrane-proximal cytoskeletal rearrangements

• Biochemical fractionation:

  • Separate G-actin and F-actin fractions using ultracentrifugation

  • Quantify IVNS1ABP distribution between fractions under various treatments

  • Perform in vitro F-actin binding/bundling assays with purified components

• Cytoskeletal perturbation experiments:

  • Latrunculin B (F-actin depolymerization) treatment at 0.5-2μM

  • Jasplakinolide (F-actin stabilization) at 50-200nM

  • Monitor IVNS1ABP relocalization and phosphorylation status

  • Assess cellular protective effects against actin destabilization-induced apoptosis

• Structure-function analysis:

  • Generate domain deletion mutants (ΔBTB, ΔKelch) to assess domain-specific functions

  • Point mutations at key residues within Kelch repeats to disrupt F-actin binding

  • Investigate binding partner interactions using proximity-dependent biotinylation (BioID)

How can researchers investigate IVNS1ABP's role in macrophage inflammatory responses?

Recent studies highlight IVNS1ABP's role in modulating macrophage responses to inflammatory stimuli. Advanced methodologies include:

• Macrophage polarization studies:

  • Monitor IVNS1ABP expression during M1 (pro-inflammatory) vs. M2 (anti-inflammatory) polarization

  • Assess cytoskeletal changes using FITC-conjugated IVNS1ABP antibodies alongside phalloidin staining

  • Quantify phagocytic capacity under IVNS1ABP overexpression/knockdown conditions

• Transcriptional regulation investigation:

  • ChIP assays to study c-myc binding to the Ivns1abp promoter under inflammatory conditions

  • Luciferase reporter assays with wild-type and mutant promoter constructs

  • Evaluate histone modifications at the Ivns1abp locus during inflammation

• Functional consequences assessment:

  • Phagocytosis assays using fluorescent beads or labeled bacteria

  • Migration/chemotaxis analysis using Transwell or Ibidi μ-slide systems

  • Cytokine production profiling via multiplex ELISA or Cytometric Bead Array

• In vivo validation approaches:

  • Generate macrophage-specific Ivns1abp knockout models

  • Challenge with inflammatory stimuli (LPS, zymosan)

  • Assess tissue inflammation and resolution kinetics

These methodologies provide comprehensive insights into IVNS1ABP's role in regulating macrophage function during inflammation.

What are the most common issues with FITC-conjugated antibodies and how can they be addressed?

For troubleshooting FITC-specific issues, use a spectral detector to confirm emission profile and rule out photoconversion or unexpected spectrum shifts .

How should researchers address discrepancies between IVNS1ABP localization patterns in different experimental systems?

When conflicting localization patterns are observed:

• Systematic validation approach:

  • Compare multiple antibody clones targeting different IVNS1ABP epitopes

  • Validate with genetic tagging (IVNS1ABP-GFP) or CRISPR knock-in of endogenous tags

  • Perform subcellular fractionation followed by Western blot as biochemical validation

• Biological variable consideration:

  • Cell cycle dependence: Synchronize cells and analyze IVNS1ABP localization at defined cell cycle stages

  • Cell type differences: Compare expression patterns across multiple relevant cell types

  • Stimulus-dependent relocalization: Standardize culture conditions and stress inducers

• Technical parameter standardization:

  • Fixation method comparison: Both paraformaldehyde and methanol fixation should be tested

  • Antibody concentration normalization: Titrate antibodies to optimal signal-to-noise ratio

  • Image acquisition settings: Use consistent exposure times and processing parameters

What experimental controls are essential for validating IVNS1ABP antibody specificity?

Rigorous validation requires multiple controls:

• Genetic controls:

  • siRNA/shRNA knockdown: ≥70% reduction in target expression

  • CRISPR/Cas9 knockout: Complete elimination of target protein

  • Rescue experiments: Re-expression of siRNA-resistant constructs

• Antibody specificity controls:

  • Peptide competition: Pre-incubation with immunizing peptide should abolish signal

  • Multiple antibodies targeting different epitopes

  • Recombinant protein controls: Overexpression systems with tagged IVNS1ABP

• Technical controls:

  • Secondary antibody-only control

  • Isotype control at equivalent concentration

  • Endogenous IVNS1ABP-negative cell line (if available)

• Functional validation:

  • Correlation of localization with known functions

  • Response to stimuli known to affect IVNS1ABP (e.g., influenza virus infection)

  • Co-localization with established interaction partners

Implementing these comprehensive controls ensures confidence in experimental findings and facilitates detection of genuine biological effects versus technical artifacts.

How can IVNS1ABP antibodies be used to investigate its role in Immunodeficiency 70?

Recent genetic data has implicated IVNS1ABP in Immunodeficiency 70, opening new research avenues:

• Patient-derived cell analysis:

  • Compare IVNS1ABP expression, localization, and phosphorylation status in patient vs. control cells

  • Assess cytoskeletal organization using FITC-conjugated IVNS1ABP antibodies

  • Evaluate immune cell functions (phagocytosis, migration, immune synapse formation)

• Variant functional characterization:

  • Generate disease-associated mutations using CRISPR base editing

  • Assess protein stability, interaction network, and subcellular localization

  • Determine impact on downstream signaling pathways

• Therapeutic exploration:

  • Screen for compounds that restore IVNS1ABP function or bypass pathways

  • Develop gene correction approaches

  • Identify compensatory mechanisms in resilient patients

• Mechanistic investigation:

  • RNA-seq to identify dysregulated pathways

  • Phosphoproteomics to map altered signaling networks

  • Interactome analysis to identify disrupted protein-protein interactions

These approaches may provide insights into disease mechanisms and potential therapeutic targets.

What novel approaches can be used to study IVNS1ABP's role in alternative splicing regulation?

Emerging technologies offer new opportunities to investigate IVNS1ABP's splicing functions:

• Transcriptome-wide approaches:

  • RIP-seq (RNA immunoprecipitation sequencing) to identify direct RNA targets

  • CLIP-seq (Cross-linking immunoprecipitation) for precise binding site mapping

  • Nanopore direct RNA sequencing to characterize full-length isoforms

• In situ visualization techniques:

  • FISH-IF co-labeling to visualize target RNAs and IVNS1ABP simultaneously

  • Live-cell imaging with MS2-tagged RNAs and fluorescently labeled IVNS1ABP

  • Proximity ligation assays to detect interactions with splicing factors

• Functional genomics screening:

  • CRISPR-Cas13 RNA targeting to modulate specific IVNS1ABP-RNA interactions

  • Splicing reporter assays with minigene constructs

  • High-throughput mutagenesis of IVNS1ABP binding sites

• Structural approaches:

  • Cryo-EM studies of IVNS1ABP-containing ribonucleoprotein complexes

  • Hydrogen-deuterium exchange mass spectrometry to map conformational changes

  • Single-molecule FRET to analyze binding dynamics

These cutting-edge approaches will provide mechanistic insights into IVNS1ABP's role in RNA processing.

How might IVNS1ABP research contribute to understanding novel antiviral mechanisms?

IVNS1ABP's interaction with influenza virus NS1 protein suggests potential antiviral applications:

• Drug discovery approaches:

  • High-throughput screens for compounds that modulate IVNS1ABP-NS1 interactions

  • Structure-based design of peptide inhibitors targeting interaction interfaces

  • Development of degraders (PROTACs) targeting viral proteins through IVNS1ABP machinery

• Broad-spectrum antiviral investigation:

  • Screen for IVNS1ABP interactions with other viral proteins

  • Compare cytoskeletal responses across multiple viral infections

  • Identify common mechanisms that could be targeted therapeutically

• Host-directed therapy exploration:

  • Modulate IVNS1ABP expression or activity to enhance intrinsic antiviral responses

  • Target downstream pathways regulated by IVNS1ABP

  • Combination approaches with direct-acting antivirals

• Predictive modeling:

  • Systems biology approaches to model IVNS1ABP network perturbations during infection

  • Machine learning to identify patterns in host response

  • Computational drug repurposing to identify existing compounds that may modulate IVNS1ABP pathways

These research directions may yield novel antiviral strategies targeting host factors rather than viral components, potentially reducing the risk of resistance development.

How can researchers combine FITC-conjugated IVNS1ABP antibodies with other molecular tools for comprehensive analysis?

Multimodal approaches enhance research depth:

• Correlative microscopy workflows:

  • CLEM (Correlative Light and Electron Microscopy): Visualize FITC-labeled IVNS1ABP followed by ultrastructural analysis

  • Combine immunofluorescence with super-resolution techniques (STORM, PALM) for nanoscale localization

  • Implement live-cell imaging followed by fixation and immunolabeling of the same cells

• Multi-omics integration:

  • Combine imaging with proteomics by using laser capture microdissection of IVNS1ABP-rich regions

  • Correlate transcriptomics data with protein localization patterns

  • Implement spatial transcriptomics in tissues with IVNS1ABP immunofluorescence

• Functional association techniques:

  • Implement optogenetic control of IVNS1ABP with simultaneous imaging

  • Combine FRET sensors for cytoskeletal dynamics with IVNS1ABP visualization

  • Use microfluidic devices for controlled stimulation during live imaging

These integrated approaches provide multidimensional data for comprehensive mechanistic insights.