HIST1H4A (Ab-16) Antibody

What is HIST1H4A and why is acetylation at Lysine 16 significant?

HIST1H4A (Histone Cluster 1, H4a) is one of several genes encoding histone H4, a core component of the nucleosome. Nucleosomes function to wrap and compact DNA into chromatin, regulating DNA accessibility to cellular machinery involved in transcription, DNA repair, replication, and maintaining chromosomal stability . The acetylation of H4 at lysine 16 (H4K16ac) represents a crucial post-translational modification that alters chromatin structure by reducing the interaction between adjacent nucleosomes, thereby promoting a more open chromatin conformation. This specific modification is implicated in diverse biological processes including transcriptional activation, DNA damage response, and cell cycle progression .

The significance of H4K16ac stems from its unique ability to prevent higher-order chromatin folding, distinguishing it from other acetylation sites on histone tails. Additionally, it serves as a binding site for proteins containing bromodomains, which recognize acetylated lysine residues and mediate downstream biological effects. Research has demonstrated that dysregulation of H4K16ac is associated with various pathological conditions, making antibodies targeting this modification valuable tools for epigenetic studies .

How do polyclonal and monoclonal HIST1H4A antibodies differ in research applications?

The distinction between polyclonal and monoclonal antibodies targeting H4K16ac significantly impacts research applications:

Polyclonal antibodies like ABIN7139167 recognize multiple epitopes around the acetylated lysine 16 site, offering high sensitivity with potential trade-offs in specificity . These are generated by immunizing animals (typically rabbits) with synthetic peptides containing the acetylated lysine 16 residue and purifying the resulting antibodies through antigen affinity methods .

Monoclonal antibodies such as EPR1004 (ab194352) provide superior consistency between experiments and batches, making them preferable for quantitative analysis . For applications requiring reproducible results over extended research periods, monoclonal antibodies offer advantages in consistency and specificity, particularly for precise mapping of H4K16ac distribution across the genome using techniques like ChIP-seq .

What validation methods confirm HIST1H4A antibody specificity?

Establishing antibody specificity is critical for reliable experimental outcomes. For H4K16ac antibodies, the following validation methods are essential:

• Peptide competition assays: Pre-incubating the antibody with the specific acetylated peptide (K16ac) should abolish signal, while pre-incubation with unmodified or differently modified peptides should not affect antibody binding .

• Immunoblotting with recombinant histones: The antibody should selectively recognize H4K16ac and not other acetylation sites (K5, K8, K12) or modifications (methylation, phosphorylation) .

• HDAC inhibitor treatment: Cells treated with histone deacetylase inhibitors (e.g., TSA) should show increased H4K16ac signal in immunofluorescence or Western blot assays, as demonstrated in validation studies with ab109463 .

• Sequential ChIP: Performing ChIP with H4K16ac antibody followed by re-ChIP with general H4 antibody can confirm the modification-specific nature of the binding .

• Knockdown of acetyltransferases: Depleting enzymes responsible for H4K16 acetylation (e.g., MOF/KAT8) should reduce signal, confirming antibody specificity .

Researchers should verify that their selected antibody has undergone these validation procedures to ensure experimental reliability and reproducibility.

What are the optimal application conditions for HIST1H4A (Ab-16) antibodies?

Different applications require specific antibody dilutions and sample preparation methods:

For ChIP applications, both polyclonal (07-329) and monoclonal (ab109463) antibodies have demonstrated efficacy in genome-wide profiling of H4K16ac distribution . The monoclonal antibody ab109463 has been validated in the CUT&RUN protocol at a concentration of 2 μg per 2.5×10^5 HeLa cells, yielding high-quality genomic profiles .

For immunofluorescence studies, optimal results require methanol-free formaldehyde fixation, with antibody incubation times of 1-2 hours at room temperature or overnight at 4°C. Nuclear counterstaining with DAPI facilitates visualization of H4K16ac enrichment patterns within nuclei .

What controls are essential when working with HIST1H4A (Ab-16) antibodies?

Implementing appropriate controls ensures reliable interpretation of experimental results:

• Positive controls:

  • Histone deacetylase inhibitor (HDACi) treated cells (e.g., TSA, sodium butyrate) show increased H4K16ac levels

  • Cell lines with known high levels of H4K16ac (HeLa cells exhibit distinct H4K16ac patterns)

  • Recombinant H4K16ac peptide for Western blot standardization

• Negative controls:

  • Isotype-matched IgG (rabbit monoclonal IgG for ab109463) for flow cytometry and ChIP experiments

  • Peptide competition to demonstrate specificity

  • Primary antibody omission controls

• Internal controls:

  • Parallel detection of total histone H4 to normalize acetylation levels

  • Housekeeping genes in ChIP-qPCR as background controls

  • Known H4K16ac-enriched genomic regions (e.g., active promoters) as ChIP positive controls

For flow cytometry applications, isotype control antibodies at matching concentrations are essential for setting appropriate gates and determining background signal levels, as demonstrated in validation studies with HeLa cells .

How should samples be prepared for optimal HIST1H4A (Ab-16) antibody detection?

Sample preparation is critical for successful detection of H4K16ac:

• For Western blotting:

  • Direct acid extraction: Incubate cell pellets in 0.2N HCl overnight at 4°C, neutralize with NaOH

  • Histone isolation kits provide cleaner preparations with reduced background

  • Use 15-18% gels to resolve the low molecular weight (11 kDa) histone H4

  • Transfer to PVDF membranes at lower voltage (30V) overnight for improved retention

• For immunofluorescence/immunocytochemistry:

  • Fix cells with 4% paraformaldehyde for 10 minutes at room temperature

  • Permeabilize with 0.1-0.5% Triton X-100 for 5-10 minutes

  • Block with 5% BSA or 10% normal goat serum with 0.3M glycine to reduce non-specific interactions

  • Counterstain nuclei with DAPI for localization reference

• For ChIP/ChIP-seq:

  • Crosslink cells with 1% formaldehyde for 10 minutes at room temperature

  • Quench with 125mM glycine

  • Lyse cells and isolate nuclei before sonication to generate 200-500bp fragments

  • Pre-clear chromatin with protein A/G beads before antibody incubation

For flow cytometry applications, fixation with 80% methanol is recommended, as demonstrated in protocols for ab109463, followed by permeabilization with 0.1% PBS-Tween for 20 minutes .

What are common problems in ChIP experiments with HIST1H4A (Ab-16) antibody and how can they be resolved?

ChIP experiments using H4K16ac antibodies may encounter several challenges:

For challenging samples, the CUT&RUN technique using pAG-MNase provides an alternative approach with higher signal-to-noise ratio and reduced background compared to traditional ChIP. This method has been validated with the monoclonal anti-H4K16ac antibody ab109463 for genome-wide profiling .

To improve specificity, sequential ChIP (performing initial ChIP with anti-H4K16ac followed by re-ChIP with another antibody of interest) can identify genomic regions with co-occurrence of different histone modifications, revealing functional relationships between epigenetic marks.

How can Western blot detection of HIST1H4A acetylation be optimized?

Optimizing Western blot protocols for H4K16ac detection requires attention to multiple factors:

• Sample preparation:

  • Acid extraction improves histone isolation

  • Add deacetylase inhibitors (5mM sodium butyrate, 1μM TSA) to lysis buffers

  • Maintain low pH during extraction to preserve acetylation marks

• Gel electrophoresis:

  • Use high percentage (15-18%) gels for optimal separation

  • Load appropriate amount (5-10μg) of histone extract

  • Include molecular weight markers below 15 kDa

• Transfer and detection:

  • PVDF membranes preferred over nitrocellulose for histone retention

  • Use BSA (not milk) for blocking to prevent non-specific binding

  • Optimize primary antibody concentration (1:200-1:2000)

  • Extended overnight incubation at 4°C improves signal

• Controls and normalization:

  • Run parallel blots with total H4 antibody for normalization

  • Include positive control (TSA-treated samples) and negative control (unmodified H4 peptide)

When troubleshooting weak signals, consider enhanced chemiluminescence (ECL) substrates with higher sensitivity or increase antibody concentration while maintaining high blocking buffer concentration (5% BSA) to preserve signal-to-noise ratio.

How do you address non-specific binding and cross-reactivity issues with histone modification antibodies?

Cross-reactivity represents a significant challenge in histone modification research:

• Pre-absorption strategies:

  • Pre-incubate the antibody with unmodified histone peptides to absorb antibodies that recognize the unmodified state

  • Perform peptide competition assays with related modifications (H4K5ac, H4K8ac, H4K12ac) to confirm specificity

• Blocking optimization:

  • Use higher concentrations of blocking agent (5% BSA)

  • Include 0.3M glycine to reduce non-specific protein-protein interactions

  • Consider specialized blocking buffers designed for histone antibodies

• Antibody selection:

  • Choose antibodies with demonstrated specificity through multiple validation methods

  • Monoclonal antibodies like EPR1004 (ab109463) typically exhibit less cross-reactivity than polyclonals

  • Verify specificity against peptide arrays containing various histone modifications

• Stringent washing:

  • Increase washing steps and duration

  • Use higher salt concentration in wash buffers for immunoprecipitation protocols

  • Include low concentrations of non-ionic detergents (0.1% Tween-20)

Dot blot arrays with modified and unmodified histone peptides provide a systematic approach to characterizing antibody cross-reactivity before proceeding with more complex applications like ChIP-seq.

How can HIST1H4A (Ab-16) antibodies be used in multi-parameter epigenetic studies?

Integrating H4K16ac analysis with other epigenetic markers yields comprehensive insights into chromatin regulation:

• Sequential ChIP approaches:

  • Perform initial ChIP with H4K16ac antibody

  • Elute and perform secondary ChIP with antibodies against other modifications

  • Quantify co-occurrence of H4K16ac with active (H3K4me3) or repressive (H3K27me3) marks

• Multi-omics integration:

  • Combine ChIP-seq using H4K16ac antibodies with:

    • RNA-seq to correlate acetylation with transcriptional output

    • ATAC-seq to assess chromatin accessibility

    • DNA methylation profiling to examine interplay between histone acetylation and DNA methylation

• Single-cell approaches:

  • Combine flow cytometry using H4K16ac antibodies with other cellular markers

  • Implement CUT&Tag protocols for single-cell epigenomic profiling with H4K16ac antibodies

• Time-course studies:

  • Monitor dynamic changes in H4K16ac during cellular differentiation or stress response

  • Correlate with changes in other epigenetic modifications and transcriptional profiles

For integrative analysis, computational approaches like ChromHMM or EpiSignature can classify genomic regions based on combinatorial patterns of H4K16ac and other epigenetic marks, revealing functional chromatin states associated with specific biological processes.

What bioinformatic approaches are recommended for analyzing HIST1H4A (Ab-16) ChIP-seq data?

Analysis of H4K16ac ChIP-seq data requires specialized bioinformatic workflows:

• Quality control and preprocessing:

  • Assess sequencing quality with FastQC

  • Filter low-quality reads and trim adapters

  • Align to reference genome with Bowtie2 or BWA

• Peak calling:

  • Use MACS2 with broad peak settings for H4K16ac

  • Parameter optimizations: --broad --broad-cutoff 0.1 --nomodel

  • Consider ChromHMM for integrative analysis with other marks

• Differential binding analysis:

  • DiffBind or HOMER for comparing H4K16ac profiles between conditions

  • DESeq2 for statistical analysis of differential enrichment

• Visualization and integration:

  • IGV or UCSC Genome Browser for visualizing H4K16ac distribution

  • deepTools for generating heatmaps and enrichment profiles

  • Integrate with gene expression data using correlation analyses

• Motif and pathway analysis:

  • HOMER or MEME for motif discovery in H4K16ac-enriched regions

  • GREAT or ChIPseeker for functional annotation

  • EnrichR for pathway analysis of genes associated with H4K16ac marks

When analyzing CUT&RUN data generated with H4K16ac antibodies, such as those validated with ab109463 , specialized pipelines like CUT&RUNTools provide enhanced peak calling accuracy and fragment size distribution analysis compared to standard ChIP-seq workflows.

How can HIST1H4A (Ab-16) antibodies be applied in therapeutic development research?

HIST1H4A antibodies contribute to therapeutic development through several research avenues:

• Target validation:

  • Identify genes regulated by H4K16ac changes in disease models

  • Correlate H4K16ac patterns with disease progression

  • Screen for compounds that modulate specific H4K16ac patterns

• Mammalian display technology:

  • Monitor epigenetic changes during antibody display selection processes

  • Implement H4K16ac detection in functional screening setups

  • Assess chromatin changes during antibody maturation in engineered cell lines

• Epigenetic drug development:

  • Screen compounds targeting H4K16ac-modifying enzymes (e.g., HDAC inhibitors)

  • Establish pharmacodynamic biomarkers based on H4K16ac levels

  • Monitor target engagement through H4K16ac analysis

• Cell therapy optimization:

  • Track H4K16ac during cellular reprogramming or differentiation

  • Optimize culture conditions based on epigenetic profiles

  • Assess persistence of epigenetic modifications in therapeutic cells

In mammalian display systems, secretion of candidate antibody clones combined with H4K16ac monitoring can provide insights into epigenetic regulation during selection processes. These systems allow for complex functional assays, enhancing the identification of therapeutic candidates with desired properties .

How are HIST1H4A (Ab-16) antibodies utilized in single-cell epigenomic analyses?

Single-cell epigenomic approaches are revolutionizing our understanding of H4K16ac dynamics:

• Single-cell technologies:

  • CUT&Tag-seq with H4K16ac antibodies enables profiling at single-cell resolution

  • Mass cytometry (CyTOF) with metal-conjugated H4K16ac antibodies allows multiplexed analysis

  • Imaging approaches combining H4K16ac antibodies with other markers reveal spatial distribution

• Analytical considerations:

  • Higher antibody concentrations may be required for single-cell applications

  • Specialized noise reduction algorithms account for technical variation

  • Normalization strategies must address cell-specific biases

• Applications:

  • Resolving cellular heterogeneity in H4K16ac patterns within tissues

  • Identifying rare cell populations with distinct epigenetic signatures

  • Tracking epigenetic changes during cell state transitions at single-cell resolution

Combining single-cell H4K16ac profiling with transcriptomic analysis through multiomics approaches provides unprecedented insights into the relationship between this specific histone modification and gene expression at the individual cell level.

What is the role of HIST1H4A (Ab-16) antibodies in studying 3D chromatin organization?

H4K16ac influences higher-order chromatin structure, making these antibodies valuable for 3D genomic studies:

• Integration with chromosome conformation capture:

  • Combine H4K16ac ChIP-seq with Hi-C data to correlate acetylation with topological domains

  • HiChIP protocols utilizing H4K16ac antibodies directly map interactions mediated by this modification

  • Investigate the role of H4K16ac in defining boundaries of chromatin domains

• Super-resolution microscopy:

  • Visualize 3D distribution of H4K16ac in nuclear space

  • Correlate with other structural proteins and histone marks

  • Track dynamic changes during cellular processes with live-cell imaging

• Liquid-liquid phase separation:

  • Investigate how H4K16ac impacts formation of chromatin compartments

  • Study the role of H4K16ac in regulating biomolecular condensates

  • Examine interplay between H4K16ac and chromatin-associated proteins

Research has demonstrated that H4K16ac depletion affects chromatin compaction and nuclear organization, suggesting potential applications in studying diseases characterized by aberrant nuclear architecture.

How do quantitative proteomics approaches incorporate HIST1H4A (Ab-16) antibodies?

Advanced proteomics techniques leverage H4K16ac antibodies for comprehensive analysis:

• Enrichment strategies:

  • Immunoprecipitation with H4K16ac antibodies followed by mass spectrometry

  • SILAC labeling to quantify differences in protein interactions with H4K16ac-enriched chromatin

  • Proximity labeling approaches to identify proteins in the vicinity of H4K16ac marks

• Cross-platform validation:

  • Correlate proteomics data with genomic approaches (ChIP-seq)

  • Verify protein interactions through co-immunoprecipitation

  • Functional validation through genetic manipulation of identified interactors

• Applications:

  • Identify readers of H4K16ac marks in different cellular contexts

  • Characterize protein complexes associated with H4K16ac-enriched chromatin

  • Map enzyme-substrate relationships in acetylation/deacetylation pathways

Quantitative proteomics combined with H4K16ac antibody-based enrichment has identified novel proteins that specifically recognize this modification, expanding our understanding of its downstream effectors in various cellular processes.