ING4 Antibody, Biotin conjugated

What is ING4 and why is it important for researchers?

ING4, also known as p29ING4, belongs to the Inhibitor of Growth (ING) family of proteins. It functions as a component of the HBO1 complex, which possesses histone H4-specific acetyltransferase activity and reduced activity toward histone H3 . This complex is responsible for the bulk of histone H4 acetylation in vivo, making ING4 crucial for epigenetic regulation. ING4 plays significant roles in modulating transcriptional pathways that regulate cell proliferation and may inhibit tumor progression . Additionally, ING4 can suppress brain tumor angiogenesis through transcriptional repression of RELA/NFKB3 target genes when complexed with RELA . In the context of immunity, ING4 negatively regulates cytokine-mediated inflammatory responses by facilitating NF-κB activation of IκB promoters . Studies with Ing4-deficient mice have revealed its importance in the innate immune response, particularly in relation to LPS treatment sensitivity .

What are the specific characteristics and storage requirements for ING4 antibody, biotin conjugated?

The biotin-conjugated ING4 antibody is typically a rabbit polyclonal antibody with human reactivity . It's supplied in liquid form containing 0.01 M PBS, pH 7.4, with preservatives such as 0.03% Proclin-300 and 50% Glycerol . For storage, it's recommended to aliquot the antibody and store at -20°C, avoiding exposure to light and repeated freeze/thaw cycles to maintain antibody integrity and performance . The antibody typically has a purity level of >95% and is purified using Protein G . Shipping usually takes 5-10 working days, and the product is intended for research use only, not for diagnostic, therapeutic, or cosmetic procedures .

How should I optimize ING4 antibody dilutions for different experimental applications?

Optimization of antibody dilutions is crucial for obtaining specific and reproducible results. For biotin-conjugated ING4 antibody, manufacturers typically recommend determining optimal dilutions empirically for each application . For unconjugated ING4 antibodies, specific dilution ranges are provided: Western Blot (1:500-1:4000), Immunohistochemistry (1:20-1:500), Immunofluorescence/Immunocytochemistry (1:10-1:100), and Immunoprecipitation (1:200-1:1000) .

When optimizing dilutions:

• Begin with the manufacturer's recommended range

• Perform a dilution series experiment using positive control samples

• Include appropriate negative controls

• Assess signal-to-noise ratio at each dilution

• Select the dilution that provides optimal specific signal with minimal background

For Western blot applications, ING4 typically appears as a band at 29-35 kDa, which corresponds to its calculated molecular weight of approximately 29-30 kDa .

What sample types and preparation techniques work best with ING4 antibodies?

Based on validation data, ING4 antibodies have been successfully tested with various sample types:

For immunohistochemistry applications with ING4 antibodies, antigen retrieval is critical. The recommended protocol involves using TE buffer at pH 9.0, though citrate buffer at pH 6.0 may serve as an alternative . For Western blotting, standard protein extraction protocols appear suitable, though specific optimizations may be necessary depending on the cellular localization and expression level of ING4 in your experimental system .

How does the biotin conjugation affect experimental design considerations?

The biotin conjugation of ING4 antibody introduces several important considerations for experimental design:

• Endogenous biotin interference: Tissues rich in endogenous biotin (like liver, kidney, brain) may produce background signal. Consider blocking endogenous biotin using avidin/biotin blocking kits before applying the biotin-conjugated antibody .

• Detection systems: Use streptavidin-conjugated detection reagents (HRP, fluorophores) that provide optimal sensitivity for your application.

• Cross-reactivity: When designing multiplex experiments, avoid using multiple biotin-conjugated antibodies simultaneously unless using sequential detection methods with thorough blocking between steps.

• Signal amplification: The biotin-streptavidin interaction allows for signal amplification, which is advantageous for detecting low-abundance targets but may require additional optimization to prevent oversaturation of high-abundance targets .

• Storage precautions: Biotin conjugates are often more sensitive to light exposure than unconjugated antibodies, necessitating appropriate storage conditions to maintain performance .

How can ING4 antibodies be used to investigate NF-κB signaling pathways?

ING4 plays a significant role in regulating NF-κB signaling, making ING4 antibodies valuable tools for investigating this pathway. Research has demonstrated that ING4 suppresses NF-κB signaling in mice and is required for proper activation of the IκBα promoter . When using ING4 antibodies to study this pathway, consider the following methodological approaches:

• Co-immunoprecipitation studies: Use ING4 antibodies to pull down protein complexes and analyze interactions with NF-κB components like RelA/p65. This approach can help determine direct protein-protein interactions between ING4 and NF-κB pathway components .

• Chromatin immunoprecipitation (ChIP): Employ ING4 antibodies to examine the recruitment of ING4 to specific promoters, particularly IκB promoters and NF-κB responsive elements. This technique has revealed that Ing4 is necessary for proper H4 acetylation of select NF-κB responsive promoters .

• Comparative studies: Compare wild-type and Ing4-null cells to investigate differences in nuclear RelA levels, NF-κB promoter binding, and cytokine production. Studies have shown that Ing4-deficient macrophages exhibit elevated levels of nuclear RelA/p65 and increased binding of RelA/p65 to relevant NF-κB promoters .

• Cytokine production analysis: Measure the impact of ING4 on cytokine production following stimulation (e.g., with LPS). Research has demonstrated that ING4 suppresses the production of some, but not all, cytokines in LPS-stimulated mice .

What controls are essential when using ING4 antibodies in epigenetic research?

When investigating ING4's role in histone acetylation and epigenetic regulation, the following controls are critical:

• Antibody specificity controls:

  • Include Ing4-null or knockdown samples to confirm antibody specificity

  • Use isotype control antibodies to assess non-specific binding

  • Include pre-absorption controls with immunizing peptide when available

• Histone modification controls:

  • Include antibodies against specific histone modifications (particularly H4 acetylation) to correlate with ING4 binding

  • Use known ING4 target and non-target regions as positive and negative controls

  • Consider temporal controls to examine dynamic recruitment of ING4 to chromatin

• HBO1 complex controls:

  • Include antibodies against other HBO1 complex components to assess co-localization

  • Use conditions that disrupt the HBO1 complex to distinguish ING4-specific versus complex-dependent effects

• Functional validation:

  • Confirm findings with complementary techniques (e.g., mass spectrometry, reporter assays)

  • Use small molecule inhibitors of histone acetyltransferases or deacetylases to manipulate the system

  • Include rescue experiments with wild-type versus mutant ING4 constructs

How can researchers distinguish between direct and indirect effects of ING4 in experimental systems?

Distinguishing direct from indirect effects of ING4 requires sophisticated experimental approaches:

• Temporal analysis: Use time-course experiments with ING4 antibodies to determine the sequence of events following stimulation. Early events (minutes to hours) are more likely to represent direct effects, while later changes may be indirect consequences .

• Domain-specific mutants: Compare cells expressing wild-type ING4 versus domain-specific mutants (e.g., HBO1 interaction domain, NF-κB binding region) to dissect functional requirements.

• Inducible systems: Employ rapidly inducible ING4 expression or degradation systems (e.g., degron-tagged ING4) to distinguish immediate from secondary effects.

• Direct target identification: Combine ING4 ChIP-seq with RNA-seq and proteomics to comprehensively identify direct genomic targets versus downstream effectors.

• Biochemical reconstitution: Use purified components in cell-free systems to test direct biochemical activities independent of cellular complexity.

How can I troubleshoot weak or non-specific signals when using ING4 antibody in experimental applications?

When encountering issues with ING4 antibody performance, consider these methodological solutions:

For weak signals:

• Increase antibody concentration within the recommended range (e.g., 1:500 instead of 1:1000 for Western blots)

• Extend primary antibody incubation time (overnight at 4°C)

• Optimize antigen retrieval for IHC applications (TE buffer pH 9.0 is recommended)

• Increase protein loading for Western blots

• Use signal enhancement systems (e.g., biotin-streptavidin amplification)

• Check antibody storage conditions and expiration date

For non-specific signals:

• Increase blocking time and concentration

• Use a different blocking agent (BSA vs. non-fat milk)

• Increase washing steps between antibody incubations

• Titrate antibody to determine optimal concentration

• Pre-absorb antibody with the immunizing peptide when available

• Include Ing4-null or knockdown controls to identify non-specific bands

For both issues:

• Verify sample integrity and protein concentration

• Confirm the expected molecular weight (29-35 kDa for ING4)

• Use fresh reagents and buffers

• Include positive control samples (e.g., HEK-293, HeLa, or C6 cells)

How should researchers interpret conflicting data between ING4 antibody results and genetic knockout models?

When faced with discrepancies between antibody-based detection and genetic models, consider these interpretative frameworks:

• Antibody specificity: Commercially available ING4 antibodies target different epitopes. Results may vary based on epitope accessibility in different experimental contexts. Compare results using antibodies targeting different regions of ING4 .

• Functional redundancy: Other ING family members may compensate for ING4 loss in knockout models. Research has shown that Ing4-null mice are viable and do not develop spontaneous tumors, suggesting potential redundancy with other ING proteins .

• Context-dependent effects: ING4's function may vary by cell type or physiological state. For example, increased promoter occupancy by RelA in Ing4-null cells increases histone H4 acetylation and activation of certain cytokines (like IL-6) but requires ING4 for activation of others (like TNF-α) .

• Post-translational modifications: Some antibodies may detect specific post-translationally modified forms of ING4, which could be absent or altered in particular experimental conditions.

• Temporal considerations: Acute depletion (antibody neutralization) versus chronic absence (genetic knockout) may yield different results due to developmental compensation.

A systematic approach combining multiple antibodies, complementary techniques (e.g., mass spectrometry), and careful controls is essential for resolving these conflicts.

What are the considerations for using ING4 antibodies in studying disease models?

When applying ING4 antibodies to disease research, particularly in cancer and inflammation models, consider these methodological approaches:

• Cancer research applications:

  • ING4 may inhibit tumor progression by modulating transcriptional pathways that regulate cell proliferation

  • ING4 can suppress brain tumor angiogenesis through transcriptional repression of RELA/NFKB3 target genes

  • Compare ING4 expression and localization between normal and tumor tissues using calibrated antibody dilutions

  • Correlate ING4 levels with histone H4 acetylation patterns at specific genomic loci

• Inflammatory disease models:

  • Ing4-null mice are highly sensitive to LPS treatment and display elevated cytokine production

  • ING4 is required for proper activation of the IκBα promoter and suppresses NF-κB signaling

  • Use ING4 antibodies to track protein levels during disease progression

  • Compare ING4 binding to inflammatory gene promoters between healthy and diseased states

• Experimental validation approaches:

  • Include appropriate disease and healthy control samples

  • Validate antibody specificity in the specific tissue/cell type being studied

  • Consider differential expression of ING4 isoforms in disease states

  • Use multiple antibodies targeting different epitopes to confirm findings

Understanding ING4's dual role in activating certain promoters while suppressing others is critical for interpreting results in complex disease models .

How are ING4 antibodies being used to explore novel epigenetic mechanisms?

ING4 antibodies are increasingly valuable for investigating complex epigenetic regulatory mechanisms:

• HBO1 complex dynamics: As a component of the HBO1 complex, ING4 contributes to histone H4-specific acetyltransferase activity . Researchers can use ING4 antibodies to track the assembly and recruitment of this complex to specific genomic loci during developmental processes or in response to environmental stimuli.

• Histone modification crosstalk: ING4 antibodies, combined with histone modification-specific antibodies, can help elucidate how ING4 facilitates crosstalk between different epigenetic marks. The HBO1 complex has reduced activity toward histone H3, suggesting specificity in its targeting .

• Transcriptional elongation: Recent research suggests that ING4-containing HBO1 complexes may be responsible for acetylation of H4 and potentially transcription elongation . Using ING4 antibodies in ChIP-seq experiments can map the distribution of ING4 along gene bodies to investigate this function.

• Target gene selectivity: ING4 is required for proper H4 acetylation of select NF-κB responsive promoters, such as TNF-α and KC, but not others, like IL-6 . Antibody-based approaches can help identify the determinants of this selectivity.

What methodological approaches can enhance the specificity and sensitivity of ING4 antibody applications?

Advanced methodological approaches to optimize ING4 antibody performance include:

• Epitope-specific validation: Validate antibody specificity using multiple approaches, including Western blot analysis with Ing4-null samples, peptide competition assays, and immunoprecipitation followed by mass spectrometry.

• Multiplexing strategies: Combine ING4 antibodies with antibodies against interacting partners (like HBO1 complex components or NF-κB proteins) in co-immunofluorescence or sequential ChIP experiments to examine co-localization and co-occupancy.

• Single-cell approaches: Adapt ING4 antibody protocols for single-cell analysis techniques like CyTOF, imaging mass cytometry, or single-cell ChIP to examine cell-to-cell variability in ING4 function.

• Proximity labeling: Use ING4 antibodies in combination with proximity labeling techniques (BioID, APEX) to identify novel interacting partners in specific cellular contexts.

• Super-resolution imaging: Optimize ING4 antibody staining for super-resolution microscopy techniques to examine the spatial organization of ING4-containing complexes at subcellular resolution.

How can researchers integrate ING4 antibody data with other omics approaches to build comprehensive biological models?

Integrating ING4 antibody-derived data with multi-omics approaches provides a systems-level understanding of ING4 function:

• Integrative genomics: Combine ING4 ChIP-seq data with RNA-seq and ATAC-seq to correlate ING4 binding with chromatin accessibility and gene expression changes. This approach can reveal how ING4-mediated H4 acetylation influences transcriptional outcomes in different biological contexts .

• Proteomics integration: Correlate ING4 immunoprecipitation-mass spectrometry data with phosphoproteomics to understand how signaling pathways modulate ING4 complex formation and function. This is particularly relevant for understanding ING4's role in NF-κB signaling .

• Network analysis: Construct interaction networks based on ING4 binding partners identified through antibody-based approaches and integrate with functional genomics screens to identify synthetic lethal relationships or functional dependencies.

• Temporal dynamics: Use time-resolved antibody-based techniques (e.g., time-course ChIP or proximity labeling) combined with kinetic transcriptomics to establish cause-effect relationships in ING4-regulated processes.

• Computational modeling: Develop predictive models of ING4 function by integrating antibody-derived binding data with structural information and machine learning approaches to predict context-specific effects of ING4 on gene regulation.

By implementing these integrated approaches, researchers can develop more comprehensive models of how ING4 coordinates epigenetic regulation, transcriptional control, and cellular responses to environmental stimuli.