ino80db Antibody

What is the INO80 complex and what is its primary function in cellular processes?

The INO80 complex is a chromatin remodeling complex with the INO80 protein serving as its catalytic ATPase subunit. This complex plays crucial roles in multiple nuclear processes including transcriptional regulation, DNA replication, and DNA repair . The INO80 protein binds DNA and, as part of the larger INO80 complex, remodels chromatin by shifting nucleosomes . This activity is essential for proper gene expression regulation and genome stability maintenance.

In research contexts, understanding INO80's function provides important insights into fundamental epigenetic regulation mechanisms. The complex has been characterized as having a DNA-binding domain and is proposed to be recruited by the YY1 transcription factor to activate certain genes .

How does ino80db differ from other INO80 complex components, particularly in zebrafish models?

The ino80db protein in zebrafish (Danio rerio) is one of several INO80 complex components with specialized functions. While the core catalytic INO80 subunit is highly conserved across species, the ino80db component represents a species-specific variant found in zebrafish .

What validated applications are available for ino80db antibodies in zebrafish research?

For zebrafish-specific ino80db antibodies, validated applications include:

The ino80db antibody from Cusabio (CSB-PA374595XA01DIL) has been specifically raised against recombinant Danio rerio ino80db protein and purified through antigen affinity methods . This makes it particularly suitable for zebrafish-specific applications where human or mouse cross-reactive antibodies might not provide accurate results.

How should I optimize immunostaining protocols when using ino80db antibodies in zebrafish embryo tissues?

When optimizing immunostaining protocols for ino80db in zebrafish embryos, consider the following methodological approach:

• Fixation optimization: Start with 4% paraformaldehyde fixation for 2-4 hours at room temperature or overnight at 4°C.

• Permeabilization: Since INO80 is a nuclear protein, thorough permeabilization is essential. Use 0.5-1% Triton X-100 in PBS for at least 30 minutes at room temperature.

• Antibody validation: Always verify antibody specificity before proceeding with full experiments. This can be done through western blotting of zebrafish embryo lysates or using morpholino knockdown controls .

• Antibody concentration: Begin with the manufacturer's recommended dilution (typically in the 1:50-1:300 range for IHC applications) and optimize based on signal-to-noise ratio.

• Signal detection: Since ino80db localizes to the nucleus, counterstain with DAPI or another nuclear marker to confirm proper nuclear localization.

• Controls: Include negative controls (secondary antibody only) and, when possible, knockdown or knockout samples to validate specificity in the zebrafish model system .

What are the critical considerations for Western blot analysis using ino80db antibodies?

When performing Western blot analysis with ino80db antibodies, researchers should consider these methodological details:

• Sample preparation: Due to the nuclear localization of ino80db, ensure complete nuclear protein extraction. A specialized nuclear extraction protocol is recommended over standard whole-cell lysate methods.

• Protein loading: Based on similar INO80 antibody applications, load 15-50 μg of protein per lane for optimal detection .

• Expected molecular weight: Verify the expected molecular weight for zebrafish ino80db protein (different from human INO80 which has a predicted band size of 176 kDa) .

• Membrane transfer: For large proteins like INO80 complex components, extend transfer time or use specialized transfer systems for large molecular weight proteins.

• Blocking conditions: 5% non-fat dry milk or BSA in TBST for 1 hour at room temperature is generally effective.

• Primary antibody incubation: Incubate with ino80db antibody at the manufacturer's recommended dilution, typically overnight at 4°C.

• Exposure time: Based on experience with other INO80 antibodies, start with 30-second exposure times for chemiluminescence detection and adjust as needed .

How can ino80db antibodies be utilized in chromatin immunoprecipitation (ChIP) experiments to study genomic localization?

While standard ChIP protocols provide a starting point, optimizing ChIP experiments with ino80db antibodies requires specific considerations:

• Chromatin preparation: Due to INO80's role in chromatin remodeling, use a two-step crosslinking approach with DSG (disuccinimidyl glutarate) followed by formaldehyde to better capture chromatin-protein interactions.

• Sonication parameters: Optimize sonication conditions to generate chromatin fragments of 200-500 bp, as INO80 complexes often regulate nucleosome positioning over specific genomic regions.

• Antibody selection: Choose antibodies that have been validated for immunoprecipitation applications. For human INO80, quantities of approximately 6 μg of antibody per mg of lysate have proven effective , though this may need adjustment for zebrafish ino80db.

• Controls: Include IgG control immunoprecipitations and, when possible, ino80db knockdown or knockout samples as negative controls .

• Data analysis: When analyzing ChIP-seq data, look for enrichment at enhancers, promoters, and regions undergoing active chromatin remodeling. Compare with datasets for other remodeling complexes to identify unique and shared targets.

What approaches should be considered for investigating ino80db's role in early zebrafish embryonic development?

Based on research findings from porcine models showing INO80's critical role in embryonic development , similar investigations in zebrafish might include:

• Temporal expression analysis: Examine ino80db expression across developmental stages using RT-qPCR and immunofluorescence staining with validated antibodies.

• Knockdown studies: Design morpholinos or CRISPR-Cas9 approaches targeting ino80db, then assess developmental phenotypes with particular attention to processes involving chromatin remodeling during embryogenesis.

• Immunofluorescence colocalization: Examine the nuclear localization patterns of ino80db in relation to specific developmental markers or during key developmental transitions.

• RNA-seq after manipulation: Similar to findings in porcine embryos, perform RNA-seq on control versus ino80db-depleted embryos to identify dysregulated genes and pathways .

• Functional rescue experiments: After knockdown, attempt rescue with wild-type or mutant ino80db to identify functional domains critical for developmental processes.

How can researchers differentiate between ino80db-specific functions and broader INO80 complex activities in zebrafish models?

Distinguishing ino80db-specific functions from the broader INO80 complex requires sophisticated experimental design:

• Comparative knockdown/knockout: Systematically target different INO80 complex components and compare phenotypes to identify subunit-specific versus core complex functions.

• Domain-specific mutations: Generate constructs with mutations in specific functional domains of ino80db for rescue experiments after knockdown.

• Protein-protein interaction studies: Use co-immunoprecipitation with the ino80db antibody followed by mass spectrometry to identify zebrafish-specific interaction partners that may differ from mammalian systems.

• Comparative ChIP-seq: Perform ChIP-seq with antibodies against different INO80 complex components to identify genomic regions where ino80db co-localizes with the core complex versus regions where it may function independently.

• Evolutionary analysis: Compare the functions of ino80db in zebrafish with homologous proteins in other model organisms to identify conserved versus species-specific roles.

What are common challenges when using ino80db antibodies, and how can researchers address inconsistent results?

Common challenges with ino80db antibodies and their solutions include:

• Low signal intensity: This may result from insufficient protein expression, ineffective extraction methods, or suboptimal antibody concentration.

  • Solution: Optimize nuclear extraction protocols specifically for zebrafish tissues; increase antibody concentration; extend incubation times; use signal enhancement systems.

• High background: This can obscure specific signals and complicate data interpretation.

  • Solution: Increase blocking stringency; reduce primary and secondary antibody concentrations; include additional washing steps; pre-absorb antibodies with non-specific proteins.

• Cross-reactivity: Some antibodies may detect related proteins within the INO80 complex.

  • Solution: Validate specificity using knockdown controls; compare results with multiple antibodies targeting different epitopes of the same protein.

• Variability between experiments: Inconsistent results between replicates can complicate interpretation.

  • Solution: Standardize all aspects of the protocol, including fixation times, antibody lots, and detection methods; include consistent positive and negative controls in each experiment.

How can researchers effectively validate the specificity of ino80db antibodies in zebrafish models?

A comprehensive validation strategy for ino80db antibodies should include:

• Western blot validation: Confirm the antibody detects a band of the expected molecular weight in zebrafish samples. Compare wild-type samples with morpholino knockdown or CRISPR knockout samples to confirm specificity.

• Peptide competition assay: Pre-incubate the antibody with the immunizing peptide before immunostaining or Western blotting. A specific antibody will show significantly reduced signal.

• Immunofluorescence patterns: Verify that the subcellular localization is consistent with expected nuclear localization, similar to findings with INO80 in other species .

• Orthogonal detection methods: Compare protein detection using alternative antibodies raised against different epitopes of the same protein or using tagged protein expression.

• Cross-species comparison: Test the antibody against samples from species with high protein sequence homology but also against negative control species with low homology to confirm specificity.

How can ino80db antibodies be combined with other techniques to study chromatin dynamics during zebrafish development?

Integrative approaches combining ino80db antibodies with other techniques include:

• ChIP-seq with ATAC-seq: Combine chromatin immunoprecipitation using ino80db antibodies with ATAC-seq to correlate ino80db binding sites with regions of accessible chromatin during developmental transitions.

• Immunofluorescence with live imaging: Use fixed-time-point immunofluorescence with ino80db antibodies to complement live imaging of fluorescently tagged nucleosomes or chromatin markers, connecting dynamic changes with ino80db localization.

• Co-immunoprecipitation with mass spectrometry: Identify developmental stage-specific interaction partners of ino80db that may regulate its function during different phases of embryogenesis.

• RNA-seq following ino80db manipulation: Similar to studies in porcine embryos , analyze transcriptome changes after ino80db knockdown to identify genes and pathways regulated by this chromatin remodeler during development.

• HiC or chromosome conformation capture: Combine with ino80db ChIP data to understand how this remodeler influences three-dimensional chromatin organization during development.

What insights can be gained by comparing INO80 function across different model organisms, and how can researchers design comparative studies?

Comparative studies of INO80 across species can reveal both conserved and divergent functions:

• Evolutionary conservation analysis: Studies in porcine models have demonstrated INO80's critical role in blastocyst development through regulation of trophectoderm epithelium permeability . Similar developmental processes can be examined in zebrafish using the specific ino80db antibody.

• Cross-species experimental design:

  • Identify orthologous developmental processes across model systems

  • Use species-specific antibodies (like ino80db for zebrafish) to examine protein localization and function

  • Compare phenotypes following gene knockdown/knockout

  • Perform cross-species rescue experiments to test functional conservation

• Comparative genomics approach:

  • Analyze INO80 binding sites across species to identify conserved regulatory elements

  • Compare transcriptional responses to INO80 depletion across species

  • Identify species-specific interaction partners that may explain divergent functions

What emerging technologies might enhance research using ino80db antibodies in the future?

Emerging technologies that could enhance ino80db antibody research include:

• CUT&Tag or CUT&RUN: These techniques offer higher signal-to-noise ratios than traditional ChIP and require fewer cells, potentially allowing chromatin profiling of specific cell populations during zebrafish development.

• Single-cell protein analysis: Emerging methods for detecting proteins in single cells could allow examination of ino80db expression heterogeneity within developing tissues.

• Proximity labeling methods: BioID or APEX2 fusions with ino80db could identify transient interaction partners in living cells that might be missed by traditional co-immunoprecipitation.

• CRISPR activation/inhibition screens: Using CRISPR-based approaches to modulate gene expression in combination with ino80db functional studies could help map genetic interaction networks.

• Cryo-electron microscopy: Structural studies of zebrafish-specific INO80 complexes using purified components could reveal species-specific architectural features that influence function.