HIST1H4A (Ab-16) Antibody

What is HIST1H4A (Ab-16) Antibody and what epitope does it recognize?

HIST1H4A (Ab-16) Antibody is a polyclonal antibody raised in rabbits that specifically recognizes the acetylated lysine 16 residue of Histone H4 (H4K16ac) in human samples. This antibody targets a peptide sequence surrounding the acetylated lysine 16 position derived from Human Histone H4 . H4K16ac is a key epigenetic mark involved in gene regulation, DNA repair, and chromatin remodeling processes . As a polyclonal IgG antibody, it offers high specificity for the acetylated form of this residue, making it valuable for studying this specific histone post-translational modification. The antibody is typically supplied in an unconjugated format suitable for multiple detection methods .

What experimental applications is HIST1H4A (Ab-16) Antibody validated for?

HIST1H4A (Ab-16) Antibody has been validated for multiple experimental techniques including:

• Enzyme-Linked Immunosorbent Assay (ELISA)

• Western Blotting (WB) with recommended dilutions of 1:200-1:2000

• Immunofluorescence (IF) with recommended dilutions of 1:50-1:200 or 1:1-1:10

• Chromatin Immunoprecipitation (ChIP)

• Immunocytochemistry (ICC) with recommended dilutions of 1:20-1:200

• Immunohistochemistry (IHC) with recommended dilutions of 1:1-1:10

For optimal results in each application, pilot experiments to determine appropriate dilutions are recommended, as the ideal concentration may vary depending on sample type, preparation methods, and detection systems employed.

How should researchers validate the specificity of HIST1H4A (Ab-16) Antibody in their experimental systems?

Validating antibody specificity is critical for ensuring reliable results. Recommended validation approaches include:

• Peptide competition assays: Pre-incubating the antibody with increasing concentrations of the immunizing peptide (acetylated H4K16 peptide) should progressively reduce signal intensity.

• Positive and negative controls:

  • Positive controls: Cell lines known to express high levels of H4K16ac (e.g., specific cancer cell lines)

  • Negative controls: Samples treated with HDAC inhibitors like sodium butyrate (NaB) or trichostatin A (TSA) will show increased H4K16 acetylation

  • Mof (H4K16 acetyltransferase) knockdown cells should show reduced H4K16ac signal

• Cross-reactivity testing: Evaluate signal against other acetylated histones, particularly other acetylated lysines on H4 (K5, K8, K12) to confirm specificity.

• Western blot evaluation: A single band at approximately 11-15 kDa corresponding to Histone H4 should be observed, with signal intensity correlating with known biological conditions that alter H4K16 acetylation.

How can HIST1H4A (Ab-16) Antibody be effectively utilized in ChIP experiments to study genome-wide H4K16ac distribution?

Chromatin Immunoprecipitation (ChIP) using HIST1H4A (Ab-16) Antibody requires careful methodological consideration:

• Optimized crosslinking: Standard formaldehyde fixation (1% for 10 minutes) works for most histone modifications, but dual crosslinking with DSG (disuccinimidyl glutarate) followed by formaldehyde may improve results for studying H4K16ac at specific genomic regions.

• Sonication parameters: Aim for chromatin fragments of 200-500bp for highest resolution. Over-sonication may destroy epitopes while under-sonication reduces ChIP efficiency.

• Antibody amount optimization:

  • Start with 2-5μg antibody per ChIP reaction

  • Include IgG control and total H4 antibody as normalization controls

• Sequential ChIP considerations: For studying co-occurrence with other modifications, sequential ChIP can be performed, with H4K16ac antibody in either the first or second IP step.

• Data analysis approaches:

  • For ChIP-seq: H4K16ac is often enriched at active promoters and enhancers

  • Compare with RNA-seq data to correlate with transcriptional activity

  • In neutrophils specifically, analyze enrichment at DNA repeats and regions associated with 50kb DNA fragments generated during early apoptosis

What is the relationship between H4K16 acetylation and cellular senescence, and how can this be studied?

Research has established a strong correlation between H4K16 hypoacetylation and cellular senescence, particularly in premature aging models:

• Experimental models to study this relationship:

  • Zmpste24-deficient mouse embryonic fibroblasts (MEFs) - a premature aging model

  • Late passage wild-type MEFs

  • Mof knockdown cells (reducing H4K16ac)

  • Cells treated with HDAC inhibitors (increasing H4K16ac)

• Methodological approach:

  • Measure H4K16ac levels by Western blot or immunofluorescence

  • Assess senescence markers simultaneously:

    • SA-β-galactosidase staining

    • p16 and p21 expression

    • SASP (Senescence-Associated Secretory Phenotype) factors

• Manipulating H4K16ac levels:

  • Decrease acetylation: siRNA knockdown of Mof (H4K16 acetyltransferase)

  • Increase acetylation: HDAC inhibitors like sodium butyrate (NaB) or trichostatin A (TSA)

• Results interpretation:

  • Mof knockdown (reducing H4K16ac) significantly increases the percentage of senescent cells

  • HDAC inhibitor treatment (increasing H4K16ac) reduces cellular senescence and improves cell survival after DNA damage

How does H4K16 acetylation influence DNA damage response, and how can HIST1H4A (Ab-16) Antibody be used to investigate this?

H4K16 acetylation plays a crucial role in DNA damage recognition and double-strand break (DSB) repair:

• Experimental design to study H4K16ac in DNA damage:

  • Induce DNA damage using ionizing radiation, etoposide, or other genotoxic agents

  • Analyze H4K16ac levels before and after damage induction using HIST1H4A (Ab-16) Antibody

  • Co-immunostaining with DNA damage markers (γH2AX, 53BP1, RAD51)

  • ChIP to determine H4K16ac enrichment at damage sites

• Key findings:

  • Basal H4K16 acetylation creates a chromatin environment conducive for DNA damage recognition

  • Mof depletion (reducing H4K16ac) delays ionizing radiation-induced focus formation of 53BP1, Rad51, MDC1, γ-H2AX, and hSSB proteins

  • Modulation of H4K16ac through Mof overexpression promotes 53BP1 recruitment to DNA damage sites in Zmpste24-deficient cells

• Recommended protocols:

  • Time-course immunofluorescence experiments using HIST1H4A (Ab-16) Antibody (1:50-1:200) following DNA damage

  • Western blot analysis of nuclear fractions at various timepoints after damage

  • ChIP-seq to identify genome-wide changes in H4K16ac distribution following DNA damage

What is the role of H4K16ac in neutrophil differentiation and programmed cell death?

H4K16ac exhibits a specialized dual role in neutrophils that can be studied using HIST1H4A (Ab-16) Antibody:

• H4K16ac patterns in neutrophils:

  • Peripheral neutrophils show massive hyperacetylation of H4K16

  • This modification is enriched at specific DNA repeats in neutrophils

  • These H4K16ac-enriched regions present accessible chromatin conformation

• Functional significance:

  • H4K16ac plays a role in regulating myeloid cell differentiation

  • H4K16ac-enriched regions are associated with cleavage sites that generate 50kb DNA fragments during early stages of programmed cell death

  • This suggests a non-canonical structural role in poising chromatin for cleavage during neutrophil apoptosis

• Experimental approach using HIST1H4A (Ab-16) Antibody:

  • ChIP-seq to map genome-wide H4K16ac distribution in neutrophils versus other blood cells

  • Correlate H4K16ac enrichment with DNase I hypersensitivity to assess chromatin accessibility

  • Compare H4K16ac patterns with DNA fragmentation patterns during neutrophil apoptosis

  • Analyze changes in H4K16ac during neutrophil differentiation from progenitor cells

What are common issues when using HIST1H4A (Ab-16) Antibody and how can they be resolved?

How can researchers best compare H4K16ac with other histone modifications using HIST1H4A (Ab-16) Antibody?

When studying the interplay between H4K16ac and other histone modifications:

• Multi-color immunofluorescence:

  • Use directly conjugated antibodies where possible to reduce cross-reactivity

  • Carefully select compatible fluorophores with minimal spectral overlap

  • Include single-staining controls to assess bleed-through

• Sequential ChIP (Re-ChIP):

  • First IP with HIST1H4A (Ab-16) Antibody

  • Elute complexes under mild conditions to preserve epitopes

  • Second IP with antibody against another histone modification

  • Include appropriate controls for each IP step

• Normalization considerations:

  • Always normalize H4K16ac to total H4 levels

  • Account for potential interdependence between modifications

  • Use mass spectrometry approaches for absolute quantification when possible

• Data analysis frameworks:

  • For genome-wide studies, develop analysis pipelines that can identify regions with co-occurrence or mutual exclusivity of modifications

  • Use pathway analysis tools to identify biological processes associated with specific modification patterns

How can HIST1H4A (Ab-16) Antibody be used to study age-related diseases and premature aging syndromes?

H4K16 hypoacetylation has been associated with premature aging conditions:

• Relevant disease models:

  • Hutchinson-Gilford Progeria Syndrome (HGPS) cells

  • Zmpste24-deficient mice (model for laminopathies)

  • Werner syndrome cells

  • Normal aging human tissues

• Experimental approach:

  • Compare H4K16ac levels using HIST1H4A (Ab-16) Antibody across disease models and healthy controls

  • Correlate H4K16ac levels with other aging markers

  • Test HDAC inhibitors for potential therapeutic effects:

    • Sodium butyrate (NaB) and trichostatin A (TSA) have shown promise in rescuing premature aging phenotypes

    • These compounds promote H4K16 acetylation and reduce cellular senescence

• Dose-response considerations for HDAC inhibitors:

  • Use lower concentrations than those used for anti-cancer applications

  • NaB and TSA promote H4K16 acetylation and reduce senescence in a dose-dependent manner

  • Monitor cellular toxicity alongside H4K16ac levels

• Potential extensions:

  • Evaluate effects of HDAC inhibitors on lifespan in animal models

  • Investigate correlation between H4K16ac and other aging-associated conditions:

    • Neurodegenerative disorders (Alzheimer's disease)

    • Age-dependent osteogenesis

    • Age-related inflammation

What insights can HIST1H4A (Ab-16) Antibody provide about the role of H4K16ac in hematopoietic disorders?

Given the important role of H4K16ac in neutrophil biology and hematopoietic cell differentiation:

• Relevant research questions:

  • How does H4K16ac distribution change in leukemias and other hematologic malignancies?

  • Is H4K16ac dysregulated in neutrophil dysfunction disorders?

  • Can manipulation of H4K16ac levels affect hematopoietic stem cell maintenance?

• Experimental approaches:

  • Compare H4K16ac patterns in normal versus malignant hematopoietic cells using HIST1H4A (Ab-16) Antibody

  • Analyze correlation between H4K16ac levels and differentiation markers

  • Investigate the relationship between H4K16ac and specific transcriptional programs in hematopoietic development

• Key considerations:

  • H4K16ac is massively hyperacetylated in peripheral neutrophils

  • This modification appears to regulate both differentiation and apoptosis in myeloid cells

  • H4K16ac may exhibit a non-canonical structural role in poising chromatin for cleavage during neutrophil cell death

What emerging technologies could enhance research using HIST1H4A (Ab-16) Antibody?

Several cutting-edge approaches could advance H4K16ac research:

• CUT&RUN and CUT&Tag:

  • More sensitive alternatives to traditional ChIP

  • Require less starting material and provide better signal-to-noise ratio

  • HIST1H4A (Ab-16) Antibody could be adapted for these techniques with appropriate protocol optimization

• Single-cell epigenomics:

  • Single-cell ATAC-seq combined with H4K16ac antibody-based approaches

  • Would reveal cell-to-cell variation in H4K16ac patterns

  • Particularly relevant for heterogeneous tissues and differentiation processes

• Live-cell imaging of H4K16ac dynamics:

  • Development of H4K16ac-specific intrabodies

  • Could enable real-time visualization of acetylation changes during cell cycle or DNA damage response

• Proteomics approaches:

  • HIST1H4A (Ab-16) Antibody could be used for affinity purification followed by mass spectrometry

  • Would identify proteins that specifically interact with H4K16-acetylated chromatin regions

How might research on H4K16ac inform therapeutic development for age-related diseases?

The close relationship between H4K16ac and cellular senescence suggests potential therapeutic applications:

• HDAC inhibitor development:

  • Current evidence shows HDAC inhibitors like NaB and TSA can promote H4K16 acetylation and reduce senescence

  • More specific HDAC inhibitors targeting relevant deacetylases could minimize off-target effects

  • Dosing strategies need to be optimized as different from cancer treatment protocols

• Targeting Mof (KAT8) activity:

  • Enhancing Mof activity could increase H4K16ac and potentially reduce senescence

  • Identifying regulators of Mof could provide additional therapeutic targets

• Monitoring efficacy:

  • HIST1H4A (Ab-16) Antibody could serve as a tool for monitoring therapy-induced changes in H4K16ac

  • H4K16ac levels could potentially serve as a biomarker for treatment efficacy

• Translational potential:

  • HDAC inhibitors have shown promise in models of:

    • Age-dependent neurodegeneration

    • Alzheimer's disease

    • Age-associated osteogenesis

    • General lifespan extension in model organisms