Acetyl-HIST1H4A (K79) Antibody

What is the Acetyl-HIST1H4A (K79) Antibody and what epitope does it recognize?

The Acetyl-HIST1H4A (K79) Antibody is a polyclonal antibody that specifically recognizes histone H4 acetylated at lysine 79. This antibody is generated using a synthetic peptide sequence surrounding the acetylated lysine 79 residue of human histone H4 as the immunogen . The antibody enables researchers to detect this specific post-translational modification, which plays important roles in chromatin dynamics and epigenetic regulation.

What are the validated applications for the Acetyl-HIST1H4A (K79) Antibody?

Based on the available data, the Acetyl-HIST1H4A (K79) Antibody has been validated for several experimental applications that are crucial for epigenetic research :

• ELISA (Enzyme-Linked Immunosorbent Assay)

• ICC (Immunocytochemistry)

• IF (Immunofluorescence)

• ChIP (Chromatin Immunoprecipitation)

These applications enable researchers to investigate the presence, distribution, and dynamics of histone H4 K79 acetylation in various experimental contexts, from protein-level detection to genome-wide localization studies.

What is the biological significance of histone H4 acetylation in chromatin regulation?

Histone H4 acetylation plays critical roles in various nuclear processes including transcriptional regulation, DNA repair, and chromatin assembly. Research has shown that regulated histone H4 acetylation is required to maintain CAG repeat stability and promote gap-induced sister chromatid recombination . In yeast, histone H4 acetylation peaks at approximately 0.6 kb from a nuclease-induced double-strand break (DSB) and can be detected up to 1.5 kb from a DSB site in mammalian cells . The dynamic nature of histone H4 acetylation, requiring both acetylation and deacetylation, rather than a particular modification state, appears crucial for preventing genomic instability, particularly at repetitive sequences .

How should researchers optimize ChIP protocols for the Acetyl-HIST1H4A (K79) Antibody?

Optimizing ChIP protocols for the Acetyl-HIST1H4A (K79) Antibody requires attention to several critical parameters:

• Crosslinking: Use 1% formaldehyde for 10-15 minutes at room temperature to effectively capture transient histone-DNA interactions

• Chromatin preparation: Sonicate to obtain fragments averaging 200-500 bp for optimal resolution

• Antibody concentration: While specific concentration is lot-dependent , a starting ratio of 2-5 μg antibody per 25-30 μg of chromatin is recommended

• Incubation conditions: Overnight incubation at 4°C with rotation provides optimal antibody binding

• Washing stringency: Include high-salt washes to reduce non-specific binding

• Controls: Always include input DNA (pre-immunoprecipitation sample), IgG control, and if possible, a known region for positive control

Studies using similar histone modification antibodies have successfully employed ChIP to detect histone H4 acetylation up to 1.5 kb from a DSB break site, demonstrating the sensitivity of this approach when properly optimized .

What validation strategies should be employed to confirm Acetyl-HIST1H4A (K79) Antibody specificity?

Rigorous validation is essential for ensuring reliable results with the Acetyl-HIST1H4A (K79) Antibody:

• Peptide competition assay: Pre-incubating the antibody with increasing concentrations of the acetylated peptide immunogen should progressively reduce signal intensity

• Comparative analysis: Test against recombinant non-acetylated H4 versus acetylated H4 to confirm specificity

• Immunoblotting controls: Similar to approaches used for other histone modification antibodies, the antibody should recognize histone H4 from eukaryotic cells but not recombinant H4 expressed in E. coli (which lacks acetylation machinery)

• Genetic controls: When possible, use cells with mutations at the K79 residue as negative controls

• Cross-reactivity testing: Examine potential cross-reactivity with other acetylated lysine residues on histone H4 using synthetic peptides

The methodology described for validating an H4 K91 acetylation antibody provides a useful model, as it demonstrated specificity by recognizing histone H4 isolated from HeLa cells but not recombinant H4 produced in E. coli .

What are critical considerations for immunofluorescence experiments using the Acetyl-HIST1H4A (K79) Antibody?

Successful immunofluorescence with the Acetyl-HIST1H4A (K79) Antibody requires careful attention to:

• Fixation method: 4% paraformaldehyde for 15 minutes preserves nuclear architecture while maintaining epitope accessibility

• Permeabilization optimization: Test different concentrations of Triton X-100 (0.1-0.5%) to ensure nuclear penetration without destroying nuclear structure

• Antigen retrieval: Consider mild citrate or EDTA-based retrieval methods if initial signals are weak

• Blocking conditions: Use 3-5% BSA or normal serum from the secondary antibody host species

• Antibody dilution: Start with manufacturer's recommendation, typically 1:100 to 1:500, and optimize as needed

• Incubation parameters: Overnight incubation at 4°C often yields better results than shorter incubations

• Nuclear counterstaining: Use DAPI at 300 nM for contrasting nuclear visualization

How should researchers analyze changes in histone H4 K79 acetylation patterns in relation to DNA damage response?

Analyzing H4 K79 acetylation in the context of DNA damage requires:

• Temporal resolution: Collect data at multiple time points following DNA damage induction (e.g., 15 min, 30 min, 1 hr, 2 hr, 4 hr) to capture dynamic changes

• Spatial distribution: Determine acetylation levels relative to damage sites using ChIP-qPCR at increasing distances from the break point

• Colocalization analysis: Perform co-immunostaining with γH2AX or other DNA damage markers to confirm association with repair foci

• Correlation with repair pathway activity: Compare acetylation patterns between cells deficient in different repair pathways

• Genetic perturbation: Analyze changes in repair efficiency when HATs or HDACs that regulate H4 acetylation are inhibited

Research has shown that histone H4 acetylation peaks at approximately 0.6 kb from a nuclease-induced DSB in yeast and extends up to 1.5 kb from a DSB in mammalian cells, suggesting a defined spatial organization of this modification during repair .

What are common pitfalls in interpreting ChIP data for histone H4 acetylation and how can they be avoided?

Researchers should be aware of several common interpretation challenges:

• Antibody cross-reactivity: Validate with appropriate controls to ensure signals represent K79 acetylation specifically

• Background signal variation: Normalize appropriately to input and IgG controls across all samples

• Cell cycle effects: Synchronize cells when possible, as histone acetylation patterns can vary throughout the cell cycle

• Signal saturation: Ensure ChIP-qPCR reactions are within the linear range of amplification

• Resolution limitations: Remember that standard ChIP has a resolution of approximately 200-500 bp, affecting precise localization claims

• Distinguishing direct from indirect effects: Complement ChIP with functional studies when making mechanistic claims

When analyzing histone H4 acetylation data in relation to DNA repair, consider that both acetylation and deacetylation appear necessary for maintaining genomic stability, suggesting a dynamic process rather than a static modification state .

How does histone H4 K79 acetylation interact with other histone modifications in nucleosome structure and function?

Understanding the interplay between H4 K79 acetylation and other modifications requires consideration of:

• Sequential ChIP analysis: Perform consecutive immunoprecipitations with antibodies against H4 K79ac and other modifications to identify co-occurring patterns

• 3D structural considerations: Examine how K79 acetylation might affect interactions with neighboring histones, as K79 is located in the globular domain rather than the N-terminal tail

• Enzyme cross-regulation: Investigate whether HATs/HDACs that modify H4 K79 also target other residues

• Modification crosstalk: Analyze how mutations or manipulations of one modification site affect the status of others

Research on other histone H4 modifications provides relevant insights. For example, studies have shown that mutation of H4 K91, another core domain residue, alters the distribution of H3 K79 methylation at telomeres, demonstrating crosstalk between modifications on different histones .

What methodological approaches can differentiate the specific functions of H4 K79 acetylation from other histone H4 acetylation sites?

To distinguish the unique roles of H4 K79 acetylation:

• Site-specific histone mutants: Generate K79 point mutations (K79A, K79R, K79Q) to abolish or mimic acetylation

• Domain-specific functional assays: Compare phenotypes of mutations in the core domain (like K79) versus N-terminal tail sites

• Temporal dynamics analysis: Compare acetylation/deacetylation kinetics between K79 and other sites following cellular perturbations

• Reader protein identification: Use peptide pull-downs with acetylated versus unacetylated K79 peptides to identify specific binding partners

• Structural stability assessments: Measure how K79 acetylation affects nucleosome or higher-order chromatin stability

Research on H4 K91 acetylation provides a useful parallel, as mutations at this core domain residue destabilize the histone octamer and lead to defects in chromatin structure, DNA repair, and transcriptional silencing .

How can researchers integrate H4 K79 acetylation data with other genomic and epigenomic datasets to gain comprehensive mechanistic insights?

For integrative analysis:

• Multi-omics correlation: Combine ChIP-seq for H4 K79ac with RNA-seq, ATAC-seq, and other histone modification ChIP-seq datasets

• Spatial genome organization: Integrate with Hi-C or similar chromatin conformation data to relate acetylation patterns to 3D genome structure

• Machine learning approaches: Apply supervised and unsupervised learning to identify patterns and associations across multiple epigenetic features

• Pathway enrichment analysis: Determine if H4 K79ac-associated genes share common biological functions or regulatory mechanisms

• Evolutionary conservation analysis: Compare H4 K79ac distribution patterns across species to identify highly conserved regulatory regions

How can cutting-edge techniques like CUT&RUN and CUT&Tag be optimized for studying histone H4 K79 acetylation?

These newer methodologies offer advantages over traditional ChIP and can be optimized for H4 K79ac studies:

• Antibody concentration optimization: Typically 1:100 to 1:200 dilution of commercial antibody, with titration experiments to determine optimal concentration

• pA-MNase/pA-Tn5 incubation parameters: 1-2 hours at 4°C for optimal binding without introducing artifacts

• Digestion or tagmentation conditions: For CUT&RUN, activate MNase with Ca²⁺ at 0°C for 30 minutes; for CUT&Tag, activate Tn5 at 37°C for 1 hour

• Cell number requirements: These methods work with fewer cells (500-5,000) compared to ChIP (millions), enabling studies with limited samples

• Signal-to-noise optimization: Include additional wash steps to reduce background

These methods provide higher resolution and lower background than traditional ChIP, potentially revealing more precise localization patterns of H4 K79 acetylation.

How might computational antibody design approaches impact future development of H4 K79 acetylation detection tools?

Recent advances in computational antibody design suggest promising directions:

• Physics- and AI-based methods: Emerging computational pipelines combine physical modeling with machine learning to improve antibody design

• Developability optimization: New approaches can simultaneously optimize binding affinity and developability characteristics like thermostability and aggregation resistance

• Epitope-specific targeting: Computational methods can enhance specificity for the exact acetylated K79 epitope while minimizing cross-reactivity

• Structural refinement: Computational approaches may help design antibodies that can better access the K79 residue in different chromatin contexts

• Humanization improvements: For mouse-derived antibodies, computational pipelines can improve humanization while maintaining specificity

Computational antibody design has already demonstrated success in creating antibodies with improved developability profiles while maintaining binding properties, as seen in recent SARS-CoV-2 antibody development .

What experimental strategies could elucidate the writers and erasers responsible for regulating H4 K79 acetylation?

To identify the enzymes controlling H4 K79 acetylation:

• HAT/HDAC inhibitor screening: Systematically test effects of specific HAT and HDAC inhibitors on H4 K79ac levels

• In vitro acetylation assays: Test candidate HATs for activity toward H4 K79 using recombinant histones and mass spectrometry

• CRISPR-Cas9 screening: Create a focused library targeting known HATs and HDACs to identify enzymes affecting H4 K79ac levels

• Proximity labeling: Use BioID or APEX2 fused to histone H4 to identify proteins associating with H4 in vivo

• Comparative analysis with known sites: Assess whether enzymes that regulate other H4 acetylation sites also impact K79

Research has identified the NuA4 complex, containing the HAT Esa1 in yeast or Tip60 in mammals, as important for histone H4 acetylation during DNA repair , making these enzymes prime candidates for investigation.