Acetyl-Histone H4 (Lys5) Antibody

What biological processes is Histone H4 Lysine 5 acetylation associated with?

Histone H4 lysine 5 acetylation is associated with multiple critical cellular processes. This modification is mediated by various histone acetyltransferase (HAT) proteins and contributes significantly to both transcriptional activation and DNA repair mechanisms. Specifically, acetylation by Esa1p in yeast or Tip60 in mammalian cells supports non-homologous end joining and replication-coupled repair pathways . Additionally, CBP/p300 family HAT proteins acetylate H4K5, functioning as transcriptional co-activators for numerous transcription factors. This modification plays a fundamental role in chromatin structure modulation, affecting gene accessibility and expression patterns .

What experimental applications are validated for Acetyl-Histone H4 (Lys5) antibodies?

Acetyl-Histone H4 (Lys5) antibodies have been rigorously validated across multiple experimental applications. The primary applications include Western Blotting (WB), Immunoprecipitation (IP), Immunohistochemistry (IHC), Immunofluorescence (IF/ICC), Chromatin Immunoprecipitation (ChIP), and ChIP-sequencing (ChIP-seq) . These antibodies demonstrate high specificity and sensitivity when used under optimized conditions. Different formats are available including polyclonal serum preparations and monoclonal antibodies, with each format offering specific advantages depending on the experimental design and research question .

What species reactivity should researchers consider when selecting an Acetyl-Histone H4 (Lys5) antibody?

When selecting an Acetyl-Histone H4 (Lys5) antibody, researchers should carefully evaluate species reactivity profiles. Most commercial antibodies demonstrate confirmed reactivity with Human, Mouse, Rat, and Monkey samples, as the antigen sequence is highly conserved across these species . For other species, many antibodies have 100% sequence homology prediction but may lack experimental validation. Researchers working with less common model organisms should evaluate sequence conservation or perform preliminary validation experiments to confirm reactivity. It's important to note that cross-reactivity validation differs between manufacturers, with some providing more extensive validation data than others .

What are the optimal dilution parameters for different experimental applications?

The optimal dilution parameters for Acetyl-Histone H4 (Lys5) antibodies vary significantly depending on the specific application and antibody format. Based on validated protocols, researchers should consider the following dilution ranges:

These parameters should be optimized for specific experimental conditions, antibody lots, and sample types .

How should researchers design ChIP experiments using Acetyl-Histone H4 (Lys5) antibodies?

When designing Chromatin Immunoprecipitation experiments with Acetyl-Histone H4 (Lys5) antibodies, researchers should implement a carefully optimized protocol. For optimal ChIP results, use 20 μl of antibody with approximately 10 μg of chromatin (equivalent to approximately 4 × 10^6 cells) per immunoprecipitation reaction . These antibodies have been validated using enzymatic chromatin IP kits, which typically provide better results than sonication-based methods for histone modifications. Researchers should include appropriate controls, such as IgG negative controls and positive controls targeting abundantly acetylated regions. For ChIP-seq applications, specialized quality control metrics should be employed to assess signal-to-noise ratios and peak distributions. Libraries should be prepared according to standard protocols with adequate sequencing depth (typically 20-30 million reads) to capture the full acetylation landscape .

What controls should be included when performing Western blot analysis of H4K5 acetylation?

Rigorous Western blot analysis of H4K5 acetylation requires several essential controls. Researchers should include both positive and negative controls to validate antibody specificity and experimental conditions. A recommended positive control is treatment of cells with histone deacetylase (HDAC) inhibitors such as sodium butyrate (10mM for 24 hours), which significantly increases global histone acetylation levels . This approach was demonstrated in HeLa cells, showing clear induction of H4K5 acetylation. Negative controls should include unmodified histone H4 or synthetic peptides lacking the acetyl modification. Additionally, researchers should run total histone H4 antibody blots in parallel to normalize acetylation signals to total histone levels, which controls for variations in chromatin extraction efficiency and loading. When comparing experimental conditions, statistical analysis should be performed on normalized values from at least three independent biological replicates .

How does H4K5 acetylation interact with other histone modifications in epigenetic regulation?

H4K5 acetylation functions within a complex network of histone modifications that collectively regulate chromatin structure and gene expression. This modification often co-occurs with acetylation at other H4 lysine residues (K8, K12, K16), creating hyperacetylated domains associated with transcriptionally active chromatin regions. Research has shown that these acetylation patterns operate within a sophisticated histone code, where combinations of modifications determine specific functional outcomes. H4K5ac positively correlates with H3K4 methylation at transcriptionally active promoters, while showing negative correlation with repressive marks like H3K27 methylation. Recent studies using genome-wide approaches have mapped the co-occurrence patterns of these modifications across different cell types and conditions, revealing context-specific regulatory mechanisms . In plants such as Arabidopsis and maize, H4K5 acetylation patterns exhibit species-specific distribution patterns that reveal evolutionary conservation and divergence of epigenetic regulation mechanisms .

What role does H4K5 acetylation play in disease states and how can researchers effectively study these contexts?

H4K5 acetylation dysregulation has been implicated in multiple disease states, particularly cancer and neurodegenerative disorders. In cancer research, altered H4K5 acetylation patterns have been observed in various tumor types, reflecting disrupted epigenetic regulation that contributes to oncogenic gene expression programs. For example, in cervical epithelial carcinoma cell lines (HeLa), H4K5 acetylation patterns show distinct nuclear localization that can be modulated by HDAC inhibitors . In neurodegenerative conditions like Friedreich ataxia, research has demonstrated altered nucleosome positioning at transcription start sites with corresponding changes in H4K5 acetylation, contributing to deficient transcriptional initiation .

To effectively study these disease contexts, researchers should employ integrative approaches combining ChIP-seq of H4K5ac with RNA-seq to correlate acetylation changes with gene expression alterations. Patient-derived samples or disease models should be compared with appropriate controls using standardized protocols to ensure reproducibility. When studying therapeutic interventions targeting histone acetylation (such as HDAC inhibitors), time-course analyses with multiple acetylation marks can reveal the dynamic epigenetic remodeling process. Advanced single-cell approaches are now being developed to characterize H4K5 acetylation heterogeneity within complex tissues, providing higher resolution understanding of disease mechanisms .

What strategies can resolve weak or inconsistent signal issues in Western blot detection of H4K5 acetylation?

Weak or inconsistent Western blot signals for H4K5 acetylation can result from multiple factors requiring systematic troubleshooting. First, researchers should optimize histone extraction protocols to ensure complete acid extraction of histones while preserving acetylation states. Using fresh extraction buffers supplemented with HDAC inhibitors (sodium butyrate, trichostatin A) is critical for preventing deacetylation during sample processing. For blotting, PVDF membranes often provide better results than nitrocellulose for histone modifications . The optimal antibody dilution should be determined empirically, typically starting at 1:1000 for Western blotting applications . Signal enhancement can be achieved using high-sensitivity ECL substrates or fluorescent secondary antibodies with digital imaging systems. When problems persist, researchers should consider alternative blocking agents (5% BSA often works better than milk for phosphorylation and acetylation modifications) and extended primary antibody incubation at 4°C overnight. Finally, loading controls should target total histone H4 rather than typical housekeeping proteins to accurately normalize for histone content variations .

How can researchers distinguish between specific signal and background in immunofluorescence studies of H4K5 acetylation?

Distinguishing specific signal from background in immunofluorescence studies of H4K5 acetylation requires careful experimental design and appropriate controls. Researchers should first optimize fixation methods, with paraformaldehyde (typically 4%) followed by permeabilization showing good results for nuclear epitopes. Antigen retrieval methods may be necessary for some sample types. The antibody dilution should be carefully titrated, with 1:800 being a recommended starting point for immunofluorescence applications . Critical controls should include: (1) secondary-only controls to assess non-specific binding of secondary antibodies; (2) peptide competition assays to confirm binding specificity; and (3) HDAC inhibitor-treated samples as positive controls showing enhanced nuclear signal .

For quantitative analysis, researchers should employ Z-stack imaging to capture the full nuclear volume and use nuclear counterstains (DAPI) to define nuclear boundaries. When comparing experimental conditions, automated image analysis pipelines can reduce observer bias and provide objective quantification of nuclear signal intensity. Advanced techniques like super-resolution microscopy can provide enhanced visualization of subnuclear distribution patterns of H4K5 acetylation, revealing association with specific chromatin domains that may not be detectable with conventional microscopy .

What factors influence ChIP-seq data quality when studying H4K5 acetylation genome-wide patterns?

Multiple technical and biological factors influence ChIP-seq data quality when profiling H4K5 acetylation patterns genome-wide. On the technical side, chromatin preparation is critical, with enzymatic digestion methods often providing better results than sonication for histone modifications. Input chromatin quality should be assessed by fragment size analysis, with optimal size ranges between 150-300bp. The antibody amount (typically 20μl per 10μg chromatin) must be optimized for each experimental system . Sequencing depth significantly impacts the detection of subtle changes, with minimum recommendations of 20 million uniquely mapped reads for point-source analysis and higher depth for broad domain detection.

Biologically, cell cycle stage dramatically affects H4K5 acetylation patterns, necessitating cell synchronization for certain comparisons. Different cell types exhibit distinct baseline acetylation landscapes that must be considered when designing cross-cell type comparisons. Environmental factors like nutrient availability can rapidly alter acetylation states, requiring careful standardization of culture conditions. For data analysis, researchers should employ specialized peak-calling algorithms optimized for histone modifications rather than transcription factor binding sites, and use appropriate normalization methods that account for global changes in acetylation levels. Integration with other genomic data types (RNA-seq, ATAC-seq, other histone modifications) provides biological context for interpreting H4K5 acetylation patterns .

How is H4K5 acetylation involved in mammalian postmeiotic sperm development?

H4K5 acetylation plays a crucial role during mammalian spermatogenesis, particularly in postmeiotic sperm development. Research by Bryant et al. characterized the dynamics of histone modifications during this process, revealing stage-specific patterns of H4K5 acetylation. During spermiogenesis, most histones are replaced by protamines, but the remaining histones retain specific modification patterns including H4K5 acetylation at developmentally important gene promoters. This epigenetic signature helps establish proper gene expression patterns required for sperm maturation and potentially influences early embryonic development after fertilization . The research employed immunofluorescence techniques to map the spatial-temporal dynamics of H4K5 acetylation throughout different stages of spermatogenesis, demonstrating progressive changes in modification patterns correlating with nuclear remodeling events. These findings suggest that H4K5 acetylation serves as an important epigenetic mark for intergenerational information transfer, potentially impacting offspring development through retained modified histones in mature sperm .

What insights have emerged from comparative genomic analyses of H4K5 acetylation patterns across species?

Comparative genomic analyses of H4K5 acetylation patterns have revealed both conserved and divergent aspects of epigenetic regulation across species. Research by He et al. conducted genome-wide chromatin immunoprecipitation studies comparing H4K5 acetylation landscapes between maize cultivars and their wild relatives . This work demonstrated that while core regulatory functions of H4K5 acetylation are conserved, significant variations exist in the distribution patterns across genomes, reflecting evolutionary adaptations in gene regulation mechanisms. The studies identified species-specific enrichment patterns around transcription start sites, enhancers, and other functional genomic elements.

Similar comparative approaches in Arabidopsis revealed that the AtEAF1 protein functions as a platform for the NuA4 acetyltransferase complex that modifies H4K5, demonstrating evolutionary conservation of acetylation machinery across plant and animal kingdoms while highlighting plant-specific adaptations . These cross-species approaches provide valuable insights into the fundamental principles of epigenetic regulation that transcend species boundaries, while also identifying lineage-specific innovations that contribute to phenotypic diversity. Methodologically, these studies require carefully standardized ChIP protocols to enable direct comparisons across species with different chromatin properties .

How does viral transformation affect global H4K5 acetylation patterns and what are the implications for disease mechanisms?

Viral transformation can profoundly reshape the global H4K5 acetylation landscape, providing insights into disease mechanisms and potential therapeutic targets. Research by Hernando et al. investigated Epstein-Barr virus (EBV)-mediated transformation of B cells, demonstrating that viral infection induces global chromatin changes independent of proliferation acquisition . Their Western blot analyses revealed significant alterations in H4K5 acetylation patterns following EBV transformation, reflecting virus-induced epigenetic reprogramming that contributes to the oncogenic process. These changes were mapped genome-wide, revealing specific regulatory regions targeted for acetylation changes that correlate with altered gene expression programs promoting cell survival and immune evasion.

Similar mechanisms have been observed with other oncogenic viruses, suggesting common epigenetic strategies exploited during viral-mediated cellular transformation. Methodologically, these studies employed chromatin immunoprecipitation coupled with next-generation sequencing (ChIP-seq) to map H4K5 acetylation changes at high resolution across the genome before and after viral infection. Integration with transcriptomic data established functional correlations between acetylation changes and gene expression alterations. These approaches not only illuminate fundamental mechanisms of viral pathogenesis but also identify potential epigenetic vulnerabilities that could be targeted therapeutically to reverse disease-associated chromatin states .