INO80 Antibody, Biotin conjugated

What is the INO80 complex and why is it important in research?

The INO80 complex is an evolutionarily conserved, ATP-dependent chromatin remodeling enzyme that plays pivotal roles in transcription regulation, DNA repair, replication, nuclear organization, and cellular differentiation. Its catalytic subunit is known as Ino80. The complex is particularly significant as it repositions nucleosomes in a DNA shape and sequence-dependent manner, facilitates histone dimer exchange, and regulates chromatin accessibility . INO80 dysregulation has been implicated in various pathological conditions, including defective heart development, arterial disease, and various cancers including melanoma, small cell lung cancer, and colon cancer .

What are the key components of the INO80 complex that antibodies might target?

The INO80 complex comprises multiple subunits, including the catalytic Ino80 subunit and several actin-related proteins. Key components that antibodies commonly target include the main INO80 protein (177 kDa) and associated proteins such as ACTR8 (also known as Arp8 or INO80N) . ACTR8 plays an important role in the functional organization of mitotic chromosomes, exhibits low basal ATPase activity, and is unable to polymerize . Other important components include Arp5, which contains domains that interact with nucleosomes and regulate INO80's enzymatic activities .

How do biotin-conjugated antibodies differ from unconjugated versions for INO80 complex research?

Biotin-conjugated antibodies offer significant advantages over unconjugated versions through their ability to interact with streptavidin-based detection systems, providing enhanced sensitivity and versatility. Unlike unconjugated antibodies (such as the INO80 antibody 24819-1-AP described in result 1, which requires secondary antibody detection), biotin-conjugated antibodies like the ACTR8 polyclonal antibody can be directly detected using streptavidin-based systems . This conjugation facilitates multi-labeling experiments, allows for signal amplification, and enables more complex experimental protocols such as chromatin immunoprecipitation sequencing (ChIP-seq) when studying INO80 complex dynamics.

How can I conduct a successful RNA immunoprecipitation (RNA-IP) experiment with INO80 antibodies?

RNA immunoprecipitation with INO80 antibodies can be performed as demonstrated in the research with linc-MYH interactions. The protocol involves:

• Prepare cell or tissue extracts (e.g., from mouse C2C12 myotubes or human myoblast-derived myocytes)

• Perform immunoprecipitation using an anti-INO80 antibody

• Extract RNA from the precipitates

• Conduct RT-PCR analysis to detect specific RNA molecules of interest

• Include appropriate negative controls (non-target RNA molecules) to verify specificity

This approach has successfully demonstrated specific interactions between INO80 and linc-MYH in both mouse and human cell systems . To enhance specificity and reduce background, optimization of washing conditions and inclusion of RNase inhibitors is recommended.

What methods can be used to visualize INO80-RNA interactions in situ?

To visualize INO80-RNA interactions within cells, researchers have successfully employed RNA in situ hybridization combined with proximity ligation assay (rISH-PLA). This technique was used to detect proximity between linc-MYH and INO80 in wild-type myoblasts. The method involved:

• Generation of a tagged version of INO80 (e.g., INO80-V5 using CRISPR-Cas9-targeted homology-directed repair)

• Isolation of muscle stem cells from appropriate animal models

• Use of biotinylated RNA-specific probes (e.g., linc-MYH-specific probes)

• Application of anti-tag antibodies (e.g., anti-V5 antibody)

• Proximity ligation to visualize close associations between the RNA and protein

This approach provides spatial information about INO80-RNA interactions that complements biochemical interaction data from co-IP and pull-down experiments.

How can I optimize binding assays to study INO80 interactions with nucleosomes?

For optimizing binding assays to study INO80-nucleosome interactions, researchers can follow this methodology:

• Prepare Cy5-labeled DNA templates (e.g., 70N5 601 DNA templates) and reconstitute nucleosomes

• Incubate increasing concentrations of INO80 complex (wild-type or mutant) with nucleosomes (25 nM) at 30°C for 30 minutes

• Use appropriate buffer conditions: 10 mM Na-HEPES (pH 7.8), 4 mM MgCl₂, 60 mM NaCl, 0.2 mM EGTA, 0.04 mM EDTA, and 8% glycerol

• Analyze reactions by resolving enzyme-bound nucleosomes from free nucleosomes on 4% native polyacrylamide gels in 1X Tris-EDTA buffer

• Repeat experiments at least three times to ensure reproducibility

This protocol allows for quantitative assessment of INO80 binding to nucleosomes and can be adapted to test different mutants or conditions.

What are common challenges when working with INO80 antibodies and how can they be addressed?

When working with INO80 antibodies, researchers commonly encounter several challenges:

• Antibody specificity: Some INO80 antibodies work well for immunoprecipitation but not for immunofluorescence . Solution: Consider generating tagged versions of INO80 (e.g., V5-tagged INO80) using CRISPR-Cas9 for improved detection.

• Protein size detection: INO80 is a large protein (calculated 177 kDa) that may show variable observed molecular weights (177 kDa, 150 kDa) . Solution: Use appropriate gel systems for large proteins and include positive controls.

• Buffer compatibility: Storage buffer contains sodium azide and glycerol which may interfere with some applications . Solution: Consider dialysis if necessary for sensitive applications.

• Temperature sensitivity: Storage recommendations (-20°C) must be strictly followed . Solution: Aliquot antibodies to avoid freeze-thaw cycles.

• Application-specific optimization: Each application (WB, IP, IHC, ChIP) requires specific dilution optimization . Solution: Perform titration experiments in each testing system to obtain optimal results.

How can INO80 antibodies be used to study chromatin remodeling mechanisms?

INO80 antibodies can be employed to investigate multiple aspects of chromatin remodeling through the following approaches:

• Chromatin Immunoprecipitation (ChIP): Using INO80 antibodies for ChIP followed by sequencing (ChIP-seq) allows genome-wide mapping of INO80 binding sites, revealing its distribution at regulatory elements such as promoters and enhancers .

• Histone variant exchange studies: Research has shown that INO80 regulates the genome-wide distribution of the histone variant H2A.Z. INO80 antibodies can be used to investigate how INO80 facilitates the replacement of nucleosomal H2A.Z/H2B with free H2A/H2B dimers .

• Proximity ligation assays: Using tagged versions of INO80 (such as INO80-V5) in combination with antibodies against other chromatin factors can reveal spatial relationships and interactions within the nuclear environment .

• In vitro remodeling assays: INO80 antibodies can be used to immunodeplete the complex from nuclear extracts to test its specific roles in ATP-dependent nucleosome remodeling reactions .

These approaches collectively provide insights into how INO80 regulates nucleosome positioning, histone variant incorporation, and chromatin accessibility.

What strategies can be used to investigate INO80's role in transcription regulation?

To investigate INO80's role in transcription regulation, researchers can employ several sophisticated strategies:

• Polymerase II stalling analysis: INO80 antibodies can be used alongside RNA Polymerase II antibodies to investigate how INO80 facilitates the release of stalled Pol II from non-coding RNA termination sites, as demonstrated in recent research .

• Transcription factor co-localization: ChIP experiments with INO80 antibodies followed by analysis of co-occupancy with transcription factors (like YY1) can reveal how INO80 influences transcription factor binding and activity .

• RNA-protein interaction studies: RNA immunoprecipitation using INO80 antibodies can identify RNAs that interact with the INO80 complex, providing insights into RNA-mediated regulation of INO80 function .

• Differential gene expression analysis: Combining INO80 ChIP-seq with RNA-seq after INO80 depletion can identify direct transcriptional targets of INO80-mediated chromatin remodeling .

These approaches reveal how INO80 couples chromatin remodeling to transcriptional regulation, RNA processing, and termination.

How can I investigate INO80's role in DNA damage repair pathways?

To investigate INO80's involvement in DNA damage repair pathways, researchers can implement the following methodological approach:

• DNA damage induction and ChIP: Treat cells with DNA damaging agents (e.g., ionizing radiation, UV, or chemical agents), then perform ChIP with INO80 antibodies to assess recruitment to damage sites.

• Co-immunoprecipitation of repair factors: Use INO80 antibodies to immunoprecipitate the complex and identify associated DNA repair proteins through mass spectrometry or western blotting.

• Repair kinetics measurement: In cells with normal or depleted INO80 levels, measure the kinetics of repair factor recruitment and resolution of DNA damage markers (γH2AX, 53BP1, etc.).

• Histone variant dynamics: INO80 facilitates homologous recombination by removing H2A.Z and promoting Rad51 filament formation . Track these histone exchange events using appropriate antibodies alongside INO80 antibodies.

• Genetic interaction studies: Combine INO80 depletion with manipulation of specific repair pathway components to identify epistatic relationships, similar to the genetic interaction studies between ino80 and htz1 .

This multi-faceted approach can elucidate INO80's specific contributions to different DNA repair mechanisms.

What experimental approaches can reveal the interplay between INO80 and long non-coding RNAs?

To investigate interactions between INO80 and long non-coding RNAs (lncRNAs), researchers can employ these methodologies:

• RNA immunoprecipitation (RNA-IP): As demonstrated with linc-MYH, use anti-INO80 antibodies to immunoprecipitate the complex and identify associated RNAs through RT-PCR or sequencing .

• RNA pull-down assays: Immobilize in vitro transcribed lncRNAs (like linc-MYH) and incubate with nuclear protein extracts, followed by mass spectrometry to identify interacting proteins. Compare results with control RNA probes to distinguish specific interactions .

• In situ visualization: Combine RNA in situ hybridization with proximity ligation assays (rISH-PLA) using INO80 antibodies to visualize RNA-protein interactions within cells .

• Functional studies: Generate lncRNA knockouts (like linc-MYH−/−) and examine effects on INO80 complex composition, chromatin association, and function. For example, research has shown that linc-MYH functions as a selective molecular switch that regulates INO80's pro-proliferative function without affecting its role in genomic stability .

These approaches collectively reveal how lncRNAs can modulate INO80 function in specific cellular contexts.

How should I analyze western blot data using INO80 antibodies?

When analyzing western blot data using INO80 antibodies, consider these technical aspects:

• Molecular weight verification: INO80 has a calculated molecular weight of 177 kDa, but may also be observed at 150 kDa. Verify that bands appear at these expected sizes .

• Positive controls: Include validated positive controls such as HeLa or HepG2 cell lysates, which have been confirmed to express detectable levels of INO80 .

• Loading controls: Use appropriate loading controls for nuclear proteins (such as Lamin B1 or histone proteins) rather than cytoplasmic controls like GAPDH.

• Quantification: For quantitative analysis, use appropriate software to measure band intensity, normalize to loading controls, and apply statistical tests to determine significance.

• Complexes vs. monomers: Consider whether high molecular weight bands might represent intact INO80 complexes, and whether sample preparation conditions might affect complex integrity.

This methodical approach ensures accurate interpretation of INO80 protein levels and modifications in experimental samples.

What are the key considerations when interpreting ChIP data for INO80 complex components?

When interpreting Chromatin Immunoprecipitation (ChIP) data for INO80 complex components, researchers should consider:

• Genomic distribution patterns: INO80 has been shown to localize to specific genomic regions, including promoters, enhancers, and DNA damage sites. Compare your distribution patterns with published datasets .

• Co-occupancy with other factors: Analyze overlap with transcription factors, histone marks, and other chromatin remodelers to understand functional relationships. For example, INO80 has been shown to interact with YY1 and WDR5 .

• Histone variant dynamics: INO80 regulates H2A.Z distribution. Consider parallel ChIP experiments for H2A.Z to understand the relationship between INO80 binding and H2A.Z localization .

• Transcriptional correlations: Correlate INO80 binding patterns with RNA Polymerase II occupancy and gene expression data to infer functional outcomes .

• Cell type specificity: Compare INO80 binding patterns across different cell types, as context-specific factors like linc-MYH can modulate INO80 function .

These considerations help researchers interpret the biological significance of INO80 binding patterns in relation to chromatin structure and gene regulation.

What controls should be included when using INO80 antibodies in immunoprecipitation experiments?

When designing immunoprecipitation experiments with INO80 antibodies, incorporate these essential controls:

• Input controls: Reserve a portion (5-10%) of the starting material prior to immunoprecipitation to verify the presence of target proteins.

• Negative antibody controls: Include an isotype-matched irrelevant antibody (e.g., rabbit IgG for rabbit polyclonal INO80 antibodies) to assess non-specific binding .

• Positive controls: If available, use tagged versions of INO80 (such as the V5-tagged INO80 generated through CRISPR-Cas9) with an anti-tag antibody as a positive control .

• Known interactors: Verify the presence of established INO80 complex components (such as ACTR8/Arp8) in the immunoprecipitate to confirm successful pulldown of the intact complex.

• Negative interactor controls: Test for proteins or RNAs that should not interact with INO80 (as demonstrated with control lncRNAs in linc-MYH studies) .

These controls ensure specificity and reliability of INO80 immunoprecipitation results.

How should I design experiments to investigate INO80's role in cell differentiation?

To investigate INO80's role in cellular differentiation, design experiments that address the following aspects:

• Temporal expression analysis: Monitor INO80 complex component expression during differentiation using western blots with INO80 antibodies. For example, in muscle differentiation, examine expression patterns in proliferating myoblasts versus quiescent muscle stem cells .

• ChIP-seq across differentiation stages: Map INO80 binding sites genome-wide at different stages of differentiation to identify dynamic binding patterns at developmental gene loci.

• Loss-of-function studies: Implement INO80 knockdown or knockout at specific stages of differentiation (using inducible systems if necessary) and assess impacts on differentiation markers and cell morphology.

• Regulatory interaction networks: Investigate interactions between INO80 and tissue-specific factors, such as the interaction between INO80 and linc-MYH in muscle cells, which regulates muscle stem cell numbers .

• In vivo models: Consider using animal models with tissue-specific INO80 manipulation, such as the INO80-V5 mice crossed with linc-MYH mutants, to assess developmental outcomes in a physiological context .