IVNS1ABP Antibody, Biotin conjugated

What is IVNS1ABP and why is it relevant in scientific research?

IVNS1ABP (Influenza Virus NS1A Binding Protein) is a protein coding gene involved in multiple cellular functions including pre-mRNA splicing, aryl hydrocarbon receptor (AHR) pathway regulation, F-actin organization, and protein ubiquitination. It plays a significant role in the dynamic organization of the actin skeleton by stabilizing actin filaments through Kelch repeats, offering protection against cell death induced by actin destabilization. In influenza research, it's particularly important as it interacts with the influenza A virus nonstructural protein NS1 and is involved in the alternative splicing of influenza A virus M1 mRNA through interaction with HNRNPK, facilitating the generation of viral M2 protein . Its multifunctional nature makes it an important target for various research applications, particularly in virology, cell biology, and cancer research.

What are the key characteristics of biotin-conjugated IVNS1ABP antibodies?

Biotin-conjugated IVNS1ABP antibodies are typically polyclonal antibodies raised in rabbits against specific amino acid sequences of the human IVNS1ABP protein. These antibodies feature biotin molecules chemically linked to the antibody structure, enabling enhanced detection when used with streptavidin-based systems. Key characteristics include:

How does epitope specificity affect experimental design when using IVNS1ABP antibodies?

The epitope specificity of IVNS1ABP antibodies significantly impacts experimental design and outcomes. Different antibodies target distinct regions of the protein: some recognize the N-terminal region (AA 1-100), while others target the C-terminal domain (AA 401-642) . This specificity affects:

• Protein Detection Efficiency: Post-translational modifications or protein interactions may mask certain epitopes but not others.

• Cross-Reactivity Profiles: Antibodies targeting highly conserved regions (like AA 47-96) show broader cross-reactivity across species (human, mouse, rat, etc.) .

• Functional Studies: When studying protein-protein interactions, antibodies targeting the interaction domain may inhibit binding, while those targeting other regions may allow detection without interference.

• Splice Variant Recognition: IVNS1ABP has multiple isoforms; epitope selection determines which variants will be detected.

For optimal experimental design, researchers should select an antibody targeting an epitope that is accessible in their experimental conditions and relevant to their research question .

What are the optimal protocols for using biotin-conjugated IVNS1ABP antibodies in ELISA?

For optimal ELISA results with biotin-conjugated IVNS1ABP antibodies, the following protocol parameters are recommended:

Sample Preparation:

• Cell/tissue lysates should be prepared in a non-denaturing buffer (typically PBS with 0.5% Triton X-100)

• Standard protein concentration: 10-20 μg/ml for cell lysates

ELISA Protocol:

• Coat plates with capture antibody (typically anti-IVNS1ABP targeting a different epitope) at 1-2 μg/ml in carbonate buffer (pH 9.6) overnight at 4°C

• Block with 2-5% BSA in PBS-T (PBS + 0.05% Tween-20) for 1-2 hours at room temperature

• Incubate samples for 2 hours at room temperature or overnight at 4°C

• Apply biotin-conjugated IVNS1ABP antibody at 1:1000-1:3000 dilution in blocking buffer for 1-2 hours

• Add streptavidin-HRP (1:5000-1:10000) for 30-60 minutes

• Develop with TMB substrate and read at 450nm

Critical Parameters:

• Antibody dilution should be optimized; starting with 1:1000 for most biotin-conjugated IVNS1ABP antibodies

• Washing steps (3-5 washes with PBS-T) between each stage are crucial for reducing background

• Temperature control during incubation affects sensitivity and specificity

How can I optimize Western blot analysis using IVNS1ABP antibodies?

While biotin-conjugated IVNS1ABP antibodies are primarily recommended for ELISA, unconjugated variants can be effectively used for Western blotting. For optimal Western blot results:

Sample Preparation:

• Lyse cells in RIPA buffer with protease inhibitors

• Load 20-40 μg of total protein per lane

Protocol Optimization:

• Transfer Conditions: For IVNS1ABP (70-72 kDa), use semi-dry transfer at 15V for 60 minutes or wet transfer at 30V overnight at 4°C

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

• Primary Antibody: Use unconjugated IVNS1ABP antibody at 1:500-1:3000 dilution overnight at 4°C

• Detection Method: If using biotin-conjugated antibody, follow with streptavidin-HRP (1:5000)

• Exposure Time: Start with 30 seconds to 5 minutes, adjust as needed

Troubleshooting Tips:

• If detecting multiple bands, increase stringency with higher dilution (1:2000-1:3000)

• The expected molecular weight is approximately 70-72 kDa

• Positive controls: Use HEK-293, HeLa, or K-562 cell lysates as confirmed positive samples

• For weak signals, increase protein loading to 50μg or extend primary antibody incubation time

What are the recommended immunohistochemistry protocols for biotin-conjugated IVNS1ABP antibodies?

For effective immunohistochemistry using biotin-conjugated IVNS1ABP antibodies:

Tissue Preparation:

• FFPE sections: 4-6 μm thickness

• Antigen retrieval: TE buffer pH 9.0 (preferred) or citrate buffer pH 6.0 at 95-98°C for 15-20 minutes

Staining Protocol:

• Deparaffinization: Standard xylene and graded alcohol series

• Endogenous Peroxidase Block: 3% H₂O₂ for 10 minutes

• Protein Block: 5% normal serum in PBS for 30 minutes

• Primary Antibody: Apply biotin-conjugated IVNS1ABP antibody (1:20-1:200 dilution) for 1-2 hours at room temperature or overnight at 4°C

• Detection: Since the antibody is already biotin-conjugated, apply streptavidin-HRP directly (1:200-1:500) for 30 minutes

• Chromogen: DAB for 5-10 minutes, monitor microscopically

• Counterstain: Hematoxylin for 30 seconds to 1 minute

Optimization Notes:

• Block endogenous biotin using avidin/biotin blocking kit before applying biotin-conjugated antibody

• Positive tissue controls: human kidney and heart tissues have shown consistent IVNS1ABP expression

• Negative controls: omit primary antibody or use isotype-matched control antibody

How should researchers account for cross-reactivity when using IVNS1ABP antibodies across different species?

Cross-reactivity considerations are crucial when designing experiments with IVNS1ABP antibodies across species:

Cross-Reactivity Data Table:

Experimental Design Strategies:

• Validation in Target Species: Always validate antibody performance in your specific species using positive controls

• Epitope Conservation Analysis: Check sequence homology for the epitope region using tools like BLAST

• Dilution Optimization: Cross-reactive antibodies often require species-specific optimization of dilution factors

• Blocking Strategy Adjustment: When working with non-human samples, consider using serum from the target species for blocking

• Alternative Detection Methods: Confirm findings with orthogonal techniques (e.g., mass spectrometry)

When working with predicted but unconfirmed cross-reactivity, preliminary validation experiments should include positive controls from the species of interest and titration of antibody dilutions to determine optimal working concentrations.

What controls should be included when conducting influenza-IVNS1ABP interaction studies?

For robust influenza-IVNS1ABP interaction studies, the following controls are essential:

Positive Controls:

• Verified Interaction Samples: Cells infected with influenza A virus with confirmed NS1-IVNS1ABP interaction

• Recombinant Protein Controls: Purified recombinant NS1 and IVNS1ABP proteins for in vitro binding assays

• Known Target Cells: NT2D1, IMR32, U87-MG, or MCF-7 cells with documented IVNS1ABP expression

Negative Controls:

• Antibody Specificity Controls:

  • Isotype-matched irrelevant antibody

  • Pre-immune serum from the same host species

  • Antibody pre-absorption with immunizing peptide

• Interaction-Specific Controls:

  • Cells infected with NS1-deficient influenza viruses

  • Cells expressing NS1 mutants that disrupt IVNS1ABP binding

  • IVNS1ABP-knockout or knockdown cells

Technical Controls:

• Input Controls: Analysis of starting material before immunoprecipitation

• Mock Infection Controls: Uninfected cells processed identically

• Time Course Controls: Samples collected at multiple time points post-infection

• Subcellular Fractionation Quality Controls: Markers for nuclear (e.g., Lamin B) and cytoplasmic (e.g., GAPDH) fractions

Implementing these controls enables reliable interpretation of results and helps distinguish specific interactions from experimental artifacts.

How can researchers effectively use biotin-conjugated IVNS1ABP antibodies in multiplex immunoassays?

Multiplexing with biotin-conjugated IVNS1ABP antibodies requires careful planning to prevent cross-reactivity and signal interference:

Multiplex Strategy Design:

• Conjugate Compatibility: When using biotin-conjugated IVNS1ABP antibodies, other antibodies in the multiplex should use different detection systems (e.g., direct fluorophore conjugates like Alexa Fluor dyes)

• Sequential Detection Protocol:

  • Apply unconjugated primary antibodies simultaneously

  • Add species-specific secondary antibodies (fluorophore-conjugated)

  • Block remaining secondary binding sites

  • Apply biotin-conjugated IVNS1ABP antibody

  • Detect with streptavidin-conjugated reporter (different fluorophore or enzyme)

Technical Optimization:

• Signal Separation:

  • For fluorescence-based detection, select fluorophores with minimal spectral overlap

  • For chromogenic detection, use distinct substrates with different colors

• Antibody Panel Design:

  Target Protein	Antibody Type	Host Species	Detection System	Order of Application
  IVNS1ABP	Biotin-conjugated	Rabbit	Streptavidin-reporter	Last
  Protein X	Unconjugated	Mouse	Anti-mouse-fluorophore	First
  Protein Y	Unconjugated	Goat	Anti-goat-fluorophore	First

• Validation Approach:

  • Test each antibody individually before combining

  • Include single-stained controls

  • Perform fluorescence minus one (FMO) controls for flow cytometry applications

This approach maximizes specificity while utilizing the high sensitivity of biotin-streptavidin detection systems.

What are common issues when using biotin-conjugated IVNS1ABP antibodies and how can they be resolved?

Researchers frequently encounter several challenges when working with biotin-conjugated IVNS1ABP antibodies:

Common Problems and Solutions:

Troubleshooting Workflow:

• Verify antibody integrity with dot blot using recombinant IVNS1ABP

• Test antibody dilution series (1:500, 1:1000, 1:2000, 1:3000)

• Modify incubation conditions (temperature, time)

• Adjust blocking reagents (BSA, normal serum, commercial blockers)

• Implement avidin/biotin blocking for tissues/cells with high endogenous biotin

How should researchers interpret complex data patterns when studying IVNS1ABP expression in influenza infection models?

Interpreting IVNS1ABP expression patterns during influenza infection requires careful data analysis and consideration of multiple factors:

Data Interpretation Framework:

• Temporal Expression Patterns:

  • IVNS1ABP expression may change at different time points post-infection

  • Compare expression at early (0-12h), middle (12-24h), and late (24-72h) infection phases

  • IVNS1ABP relocalization to nuclear bodies often occurs 4-8 hours post-infection

• Subcellular Localization Analysis:

  • Uninfected cells: IVNS1ABP is predominantly diffuse in the nucleoplasm

  • Early infection: Partial colocalization with NS1 protein

  • Late infection: Formation of distinct nuclear bodies containing both proteins

  • Quantify colocalization using Pearson's correlation coefficient or Manders' overlap coefficient

• Virus Strain-Specific Variations:

  • Different influenza subtypes show variable NS1-IVNS1ABP interaction patterns

  • H1N1 strains may show stronger interactions than H3N2 or influenza B

  • NS1 protein polymorphisms can affect binding affinity to IVNS1ABP

• Cell Type-Dependent Expression:

  • Basal IVNS1ABP expression varies between cell types:

    • Higher in epithelial cells and immune cells

    • Lower in fibroblasts and endothelial cells

  • Infection-induced changes should be normalized to cell-type-specific baselines

• Interaction with RNA Processing Machinery:

  • IVNS1ABP functions in mRNA splicing and export

  • During infection, analyze colocalization with splicing factors (e.g., HNRNPK)

  • Quantify changes in splicing patterns of viral and host targets

When conflicting data patterns emerge, consider using multiple detection methods (immunoblotting, immunofluorescence, and RT-qPCR) and focusing on consistent trends rather than absolute values.

What statistical approaches are most appropriate for analyzing IVNS1ABP quantification data?

Selecting appropriate statistical methods for IVNS1ABP quantification depends on the experimental design and data characteristics:

Recommended Statistical Approaches:

Data Normalization Strategies:

• Normalize to housekeeping proteins (GAPDH, β-actin) for Western blots

• Use total protein normalization methods (Ponceau S, REVERT stain) for more accurate quantification

• For immunofluorescence, normalize to nuclear stain or cell area

• For ELISA, use standard curves with recombinant IVNS1ABP protein

Sample Size Determination:

• Power analysis should be performed to determine minimum sample size

• For typical IVNS1ABP expression studies, n=3-5 biological replicates with 2-3 technical replicates is often sufficient to detect 50% changes with 80% power

How can biotin-conjugated IVNS1ABP antibodies be utilized in studies of virus-host protein interaction networks?

Biotin-conjugated IVNS1ABP antibodies offer several advantages for mapping complex virus-host protein interaction networks:

Advanced Applications:

• Proximity Labeling Proteomics:

  • Use biotin-conjugated IVNS1ABP antibodies in combination with crosslinking agents to capture transient interaction partners

  • Apply to influenza-infected cells at different time points

  • Identify interaction partners via streptavidin pulldown followed by mass spectrometry

  • This approach can reveal dynamic changes in the IVNS1ABP interactome during infection progression

• ChIP-Seq and RIP-Seq Applications:

  • IVNS1ABP is involved in RNA processing and binds to specific RNA motifs

  • Biotin-conjugated antibodies can be used for RNA immunoprecipitation followed by sequencing (RIP-Seq)

  • Identify direct RNA targets of IVNS1ABP during viral infection

  • Compare RNA binding profiles between uninfected and infected states

• Live-Cell Imaging with Secondary Detection:

  • Use biotin-conjugated IVNS1ABP antibodies with cell-permeable streptavidin-fluorophore conjugates

  • For permeabilized cells, track IVNS1ABP dynamics during viral infection

  • Combine with fluorescently tagged viral proteins for colocalization studies

  • Quantify protein movement and interaction kinetics

• Multiplex Interactome Analysis:

  • Combine with other antibodies to simultaneously track multiple components of the influenza-host interaction network

  • Map temporal changes in protein complex formation

  • Identify key nodes in the virus-host protein network that could serve as therapeutic targets

These advanced applications leverage the specificity of IVNS1ABP antibodies and the high-affinity biotin-streptavidin interaction to provide deeper insights into virus-host protein interactions.

What are the cutting-edge approaches for studying IVNS1ABP's role in alternative splicing during viral infection?

Investigating IVNS1ABP's role in alternative splicing during viral infection requires sophisticated methodological approaches:

Innovative Methodologies:

• CLIP-Seq (Cross-Linking Immunoprecipitation-Sequencing):

  • UV crosslink RNA-protein complexes in infected cells

  • Immunoprecipitate with biotin-conjugated IVNS1ABP antibodies

  • Sequence bound RNAs to identify direct binding sites

  • Compare binding patterns between uninfected and infected cells

  • This reveals direct RNA targets of IVNS1ABP and how they change during infection

• Splicing-Sensitive RNA-Seq:

  • Design experiments comparing wild-type cells to IVNS1ABP-depleted cells

  • Use junction-spanning primers to quantify specific splicing events

  • Apply computational tools like rMATS or MAJIQ for comprehensive alternative splicing analysis

  • Focus on viral transcripts (particularly M1/M2) and host immune response genes

• In vitro Splicing Assays:

  • Construct minigene systems containing influenza A virus splicing elements

  • Add recombinant IVNS1ABP or immunoprecipitated complexes

  • Measure splicing efficiency with varying concentrations of IVNS1ABP

  • Test the effect of NS1 protein addition on IVNS1ABP-mediated splicing

• CRISPR-Cas9 Domain Mapping:

  • Generate IVNS1ABP mutants lacking specific functional domains

  • Assess the impact on influenza viral RNA splicing

  • Quantify M1/M2 ratio changes

  • Identify critical domains required for splicing regulation

  • Combine with structural biology approaches (e.g., cryo-EM) to understand mechanism

• Single-Molecule RNA Fluorescence In Situ Hybridization (smFISH):

  • Design probes specific to differentially spliced isoforms

  • Combine with immunofluorescence for IVNS1ABP

  • Visualize and quantify splicing changes at the single-cell level

  • Track spatial and temporal dynamics of splicing regulation during infection

These approaches provide complementary information about IVNS1ABP's mechanistic role in regulating alternative splicing during viral infection.

How can researchers leverage IVNS1ABP antibodies to develop novel antiviral strategies targeting virus-host interactions?

Developing antivirals based on IVNS1ABP-NS1 interactions represents an emerging therapeutic strategy:

Translational Research Approaches:

• High-throughput Screening Platforms:

  • Develop ELISA-based assays using biotin-conjugated IVNS1ABP antibodies

  • Screen compound libraries for molecules that disrupt NS1-IVNS1ABP interaction

  • Use competition assays with known binding peptides as positive controls

  • Establish dose-response relationships for promising compounds

• Peptide-Based Inhibitor Development:

  • Map the precise binding interface between NS1 and IVNS1ABP using deletion mutants

  • Design peptide mimetics that compete for binding

  • Optimize lead peptides for stability, cell penetration, and affinity

  • Test peptide inhibitors in cellular infection models

  • Validated binding regions include amino acids 401-642 of IVNS1ABP

• Antibody-Based Therapeutic Strategies:

  • Convert research-grade antibodies into potential therapeutics

  • Engineer antibody fragments (Fab, scFv) targeting the NS1-binding domain of IVNS1ABP

  • Design bispecific antibodies targeting both viral and host proteins

  • Evaluate efficacy in cellular and animal models of influenza infection

• Structure-Based Drug Design:

  • Use structural information about IVNS1ABP-NS1 interaction

  • Perform in silico screening for small molecule binding pockets

  • Design compounds that allosterically alter IVNS1ABP conformation

  • Test candidates for disruption of splicing regulation and viral replication

• Cell-Based Phenotypic Assays:

  • Develop reporter systems for IVNS1ABP-dependent splicing events

  • Screen for compounds that modulate splicing patterns similar to IVNS1ABP depletion

  • Validate hits using biotin-conjugated antibodies to assess target engagement

  • Evaluate effects on viral replication and host cell viability

These translational approaches leverage the specificity of biotin-conjugated IVNS1ABP antibodies to bridge basic research findings and therapeutic development.

Comprehensive comparison of commercially available IVNS1ABP antibodies and their applications

IVNS1ABP protein information and functional domains

Recommended positive controls for IVNS1ABP expression studies