HIST1H4A (Ab-5) Antibody

What is HIST1H4A (Ab-5) Antibody and what epitope does it recognize?

HIST1H4A (Ab-5) Polyclonal Antibody is a rabbit-derived antibody that specifically recognizes the peptide sequence around the site of Lysine 5 derived from Human Histone H4 . This antibody targets histone H4 protein (accession number P62805) which is one of the core histone proteins comprising the nucleosome . The "(Ab-5)" designation specifically indicates that this antibody targets the region containing the lysine 5 residue of histone H4, a site that can undergo acetylation as a post-translational modification critical for regulating chromatin structure and gene expression .

What applications is HIST1H4A (Ab-5) Antibody validated for?

The HIST1H4A (Ab-5) Polyclonal Antibody has been validated for multiple experimental applications commonly used in epigenetic and chromatin research:

The antibody shows specific nuclear staining in immunofluorescence applications, consistent with the known nuclear localization of histone proteins .

How does HIST1H4A (Ab-5) Antibody compare to antibodies detecting acetylated histone H4?

While HIST1H4A (Ab-5) Antibody recognizes the region around lysine 5 regardless of modification status, antibodies specifically against acetylated H4K5 (such as those described in search result ) detect only the acetylated form of this residue. The acetylation of H4K5 is particularly significant as:

• H4K5 acetylation is catalyzed by the Hat1-RbAp46 complex

• This modification is prominent on newly synthesized histones during DNA replication

• It plays a critical role in nucleosome assembly pathways

• The modification can be experimentally induced using histone deacetylase inhibitors like sodium butyrate

When designing experiments to study histone modifications, researchers should select antibodies based on whether they wish to detect total H4 protein or specifically its acetylated form at K5.

What controls should be included when using HIST1H4A (Ab-5) Antibody in experiments?

Proper experimental controls are essential when working with histone antibodies:

For validation of acetylation-specific antibodies, treatment of cells with histone deacetylase inhibitors like sodium butyrate can increase the signal, as demonstrated in the R&D Systems documentation for H4K5ac antibodies .

How should I optimize immunofluorescence protocols for HIST1H4A (Ab-5) Antibody?

Successful immunofluorescence with histone antibodies requires careful optimization:

• Fixation:

  • 4% paraformaldehyde (10-15 minutes) typically preserves nuclear architecture

  • Methanol fixation may improve access to some nuclear epitopes

  • Test multiple fixation methods with your specific cell type

• Permeabilization:

  • Include adequate permeabilization (0.1-0.5% Triton X-100) to ensure nuclear access

  • Overly harsh permeabilization may extract nuclear proteins

• Antibody concentration:

  • Start with recommended dilution (0.1 μg/mL has been successful for similar antibodies)

  • Titrate to determine optimal signal-to-noise ratio

• Signal detection:

  • Nuclear counterstain (DAPI) helps confirm proper nuclear localization

  • Fluorophore selection should avoid spectral overlap with other channels

  • Confocal microscopy may provide better resolution of nuclear structures

• Pattern interpretation:

  • Expect nuclear localization with possible enrichment in euchromatic or heterochromatic regions

  • Different cell types may show variation in staining patterns

How can ChIP protocols be optimized for HIST1H4A (Ab-5) Antibody?

Chromatin immunoprecipitation (ChIP) with histone antibodies requires specific considerations:

• Crosslinking conditions:

  • Standard 1% formaldehyde for 10 minutes is typically sufficient

  • Over-crosslinking can mask epitopes and reduce efficiency

• Sonication parameters:

  • Aim for fragments between 200-500 bp

  • Verify fragmentation by agarose gel before proceeding

• Antibody amount:

  • Typically 2-5 μg per ChIP reaction

  • Perform antibody titration to determine optimal amount

• Washing stringency:

  • Balance between reducing background and maintaining specific interactions

  • Include high-salt washes to reduce non-specific binding

• Controls for ChIP experiments:

  • Input sample (pre-immunoprecipitation)

  • IgG control from same species as primary antibody

  • Positive control loci (housekeeping genes for active marks)

  • Negative control loci (silenced genes or heterochromatic regions)

• Validation approaches:

  • qPCR of known target regions before proceeding to sequencing

  • Compare enrichment profiles with published datasets for H4K5ac

How can HIST1H4A (Ab-5) Antibody be used to study nucleosome assembly during DNA replication?

Histone H4 acetylation at lysine 5 plays a critical role in replication-coupled nucleosome assembly:

• Experimental design approaches:

  • Synchronize cells and collect at different points in S phase

  • Combine with EdU labeling to identify actively replicating regions

  • ChIP-seq to map genome-wide distribution during replication

• Mechanistic insights:

  • Newly synthesized H4 is acetylated at K5 and K12 by the HAT1 holoenzyme (Hat1-RbAp46)

  • This pattern facilitates interaction with histone chaperones during nucleosome assembly

  • Hat1-RbAp46 acetylates H4 in H3.1-H4 complexes more efficiently than in H3.3-H4 complexes

• Analysis considerations:

  • Compare patterns in early vs. late replicating chromatin domains

  • Assess correlation with other replication-associated histone marks

  • Evaluate changes in different cell types or developmental stages

This research direction can provide insights into how chromatin states are propagated through cell division, as Hat1-RbAp46-mediated acetylation impacts nucleosome assembly differently for replication-dependent H3.1 versus replication-independent H3.3 histone variants .

How does H4K5 acetylation interact with other histone modifications in the histone code?

H4K5 acetylation functions within a complex network of histone modifications:

Understanding these interactions can provide insights into the complex regulatory mechanisms that govern chromatin structure and function.

What role does H4K5 acetylation play in the differential assembly of histone variants?

Recent research has revealed interesting connections between H4K5 acetylation and histone variant deposition:

• Variant-specific interactions:

  • Hat1-RbAp46 acetylates H4 in H3.1-H4 complexes more efficiently than in H3.3-H4 complexes

  • More Hat1 and RbAp46 co-purify with H3.1 than with H3.3

  • H4K5,12ac enrichment on H4 that co-purifies with H3.1 compared to H3.3

• Assembly pathway differences:

  • H3.1-containing nucleosomes are primarily assembled during DNA replication

  • H3.3-containing nucleosomes are assembled in a replication-independent manner

  • Different chaperone systems: CAF-1 for H3.1 versus HIRA for H3.3

• Experimental approaches:

  • Co-immunoprecipitation to study interactions between modified H4 and chaperones

  • Pulse-chase experiments to track newly synthesized histones

  • FRAP (Fluorescence Recovery After Photobleaching) to measure dynamics

• Functional implications:

  • Different acetylation patterns may help direct histones to appropriate assembly pathways

  • May contribute to maintaining distinct functions of histone variants

  • Could influence epigenetic inheritance through cell division

This differential acetylation pattern suggests a mechanistic link between histone modification and the choice of assembly pathway, potentially contributing to the establishment and maintenance of chromatin domains .

Why might Western blots with HIST1H4A (Ab-5) Antibody show unexpected bands?

Multiple or unexpected bands in Western blots can occur for several reasons:

• Common causes of multiple bands:

• Validation approaches:

  • The primary H4 band should appear at approximately 12 kDa

  • Use recombinant H4 protein as a size control

  • Perform peptide competition assays to identify specific bands

  • Compare with other validated H4 antibodies

• Technical optimization:

  • Use high percentage gels (15-18%) for better resolution of histone proteins

  • Optimize transfer conditions for small proteins

  • Consider specialized histone extraction protocols

When optimizing Western blot conditions, the R&D Systems documentation notes successful detection using PVDF membrane under reducing conditions with their specific buffer system .

How should variations in nuclear staining patterns be interpreted?

Nuclear staining patterns can provide insights into chromatin organization and histone distribution:

• Common patterns and their interpretation:

  • Even nuclear distribution: typical for many histone modifications

  • Peripheral enrichment: seen with certain histone variants (H1.2, H1.3, H1.5)

  • Punctate pattern: may indicate enrichment in heterochromatic regions

  • Exclusion from nucleoli: common for many histone marks

• Cell-type specific considerations:

  • Different cell types may show variant-specific distribution patterns

  • Cell lines lacking certain histone variants may show compensatory distribution of others

  • Compare with known nuclear architecture markers

• Technical factors affecting pattern interpretation:

  • Fixation method can alter nuclear architecture

  • Antibody concentration affects signal-to-noise ratio

  • Imaging parameters (exposure, contrast) influence pattern visibility

  • Deconvolution may improve resolution of nuclear structures

• Validation approaches:

  • Co-staining with markers of nuclear domains (heterochromatin, nucleoli)

  • Comparison with other histone modification antibodies

  • Correlation with DAPI intensity (indicates DNA density)

Research has shown that certain histone variants maintain consistent nuclear distribution patterns across different cell types, suggesting fundamental roles in nuclear organization .

How can ChIP-seq data quality with HIST1H4A (Ab-5) Antibody be assessed?

Quality assessment of ChIP-seq data is critical for valid biological interpretation:

• Key quality metrics:

  • Signal-to-noise ratio (enrichment over background)

  • Peak shape characteristics (sharp vs. broad)

  • Correlation with known genomic features

  • Reproducibility between replicates

• Computational analysis considerations:

  • Appropriate peak calling algorithms for histone modifications (typically broad)

  • Normalization methods accounting for sequencing depth

  • Comparison to input or IgG control samples

  • Visualization of coverage at known positive and negative regions

• Biological validation approaches:

  • Correlation with gene expression data

  • Comparison with published datasets for similar modifications

  • Verification of enrichment at expected genomic features

  • qPCR validation of selected loci

• Common pitfalls and solutions:

  • Low enrichment: optimize antibody amount, chromatin amount, wash conditions

  • High background: increase wash stringency, optimize antibody concentration

  • Poor reproducibility: standardize protocol, increase replicate number

  • Unexpected distribution: verify antibody specificity, consider cell type differences

Proper quality assessment ensures that downstream biological interpretations are based on reliable data, particularly important when studying histone modifications that may have subtle genomic distributions.

How are new technologies enhancing our understanding of H4K5 acetylation dynamics?

Recent technological advances are providing unprecedented insights into histone modifications:

• Single-cell approaches:

  • Single-cell ChIP-seq and CUT&Tag for cellular heterogeneity analysis

  • Single-molecule imaging to track modification dynamics in living cells

  • Integration with single-cell transcriptomics

• Genome editing applications:

  • CRISPR-based histone modification mapping

  • Creation of acetylation-mimetic or deficient histone mutants

  • Site-specific introduction of modified histones

• Structural biology insights:

  • Cryo-EM structures of nucleosomes with modified histones

  • Understanding how modifications alter nucleosome structure

  • Visualization of histone-chaperone interactions

• Quantitative proteomics:

  • PTM-specific quantification using mass spectrometry

  • Multiplexed analysis of modification combinations

  • Temporal dynamics during cell cycle progression

These technologies are driving new understanding of how H4K5 acetylation contributes to chromatin regulation and nucleosome assembly pathways.

What are emerging concepts regarding H4K5 acetylation in disease contexts?

Understanding histone modifications in disease contexts is an active area of research:

• Cancer biology implications:

  • Altered histone modification patterns in various malignancies

  • Potential diagnostic or prognostic biomarkers

  • Therapeutic targeting of enzymes regulating H4K5 acetylation

• Neurodegenerative disease connections:

  • Disrupted histone acetylation in neurodegenerative disorders

  • HDAC inhibitors as potential therapeutic agents

  • Role in neuronal gene expression regulation

• Developmental disorders:

  • Importance in embryonic development and cellular differentiation

  • Mutations in histone modifying enzymes in congenital disorders

  • Transgenerational epigenetic inheritance considerations

• Research methodologies:

  • Patient-derived samples for modification profiling

  • Disease models to study dynamic changes

  • Integration with genetic and genomic data

This research area holds promise for identifying new biomarkers and therapeutic targets based on histone modification patterns.