HIST1H4A (Ab-59) Antibody

What is HIST1H4A (Ab-59) Antibody and what cellular targets does it recognize?

HIST1H4A (Ab-59) Antibody is a rabbit polyclonal antibody that specifically recognizes human Histone H4 protein at or around the lysine 59 position. Histone H4 is a core component of nucleosomes, which wrap and compact DNA into chromatin, thus limiting DNA accessibility to cellular machinery. Histones play central roles in transcription regulation, DNA repair, DNA replication, and chromosomal stability . This antibody is typically generated using a synthetic peptide sequence around site of Lysine 59 derived from Human Histone H4 as the immunogen . The specificity for this region makes it valuable for studying specific post-translational modifications and structural aspects of histone H4.

What are the validated applications for HIST1H4A (Ab-59) Antibody?

The HIST1H4A (Ab-59) Antibody has been validated for multiple research applications:

The antibody demonstrates specific nuclear and chromosomal localization in immunostaining applications, consistent with the expected subcellular distribution of histone H4 .

What experimental controls should be included when using HIST1H4A (Ab-59) Antibody?

When designing experiments with HIST1H4A (Ab-59) Antibody, researchers should implement the following controls:

• Positive control: HeLa acid extract has been validated as an appropriate positive control for Histone H4 antibody detection .

• Negative controls: Include samples without primary antibody and use of isotype-matched non-specific antibodies.

• Peptide competition assay: Pre-incubation of the antibody with the immunizing peptide can validate specificity.

• Cross-reactivity assessments: While the antibody is designed for human samples, validation against other species should be performed if using in non-human models .

For applications requiring quantitative analysis, standard curves using recombinant proteins or synthetic peptides representing known concentrations of the target should be included .

How can researchers optimize HIST1H4A (Ab-59) Antibody for chromatin immunoprecipitation (ChIP) studies?

For optimal results in ChIP experiments using HIST1H4A (Ab-59) Antibody:

• Crosslinking optimization: For histone H4 studies, a 1% formaldehyde treatment for 10 minutes at room temperature is typically sufficient, but optimization may be needed based on cell type .

• Sonication parameters: Chromatin should be sheared to fragments of 200-500 bp for optimal immunoprecipitation. Verification of fragment size by agarose gel electrophoresis is recommended before proceeding .

• Antibody amount: Researchers should use 2-10 μl per ChIP reaction as recommended in validation protocols . This should be optimized for each experimental system.

• Washing stringency: Implement a high salt washing step (500 mM NaCl) to reduce non-specific binding while maintaining specific interactions .

• Controls: Include a no-antibody control and preferably an IgG isotype control to assess background levels .

When analyzing ChIP-Seq data with this antibody, researchers should consider histone H4's widespread distribution across the genome and focus on enrichment patterns rather than simple presence/absence .

What considerations are important when investigating histone H4 modifications in aging research with this antibody?

Recent research highlights significant changes in histone profiles during aging processes that can be studied using HIST1H4A (Ab-59) Antibody:

• Experimental design considerations:

  • Include age-matched controls when studying histone modifications in aging models

  • Consider both replicative aging and chronological aging paradigms as they show different histone profiles

  • Account for cell-type specific effects, as histone depletion patterns differ between actively replicating cells and quiescent cells

• Key findings in aging research:

  • Studies demonstrate age-related histone H4 loss in various models, with reductions of up to 47% in late passage human diploid fibroblasts

  • Global downregulation of histones, including H4, has been observed in aged T cells, leading to delayed S-phase progression

  • Both actively proliferating and postmitotic cells develop histone loss with aging, though patterns may differ

• Methodological approach:

  • When studying aging effects, combine immunoblotting with mass spectrometry to distinguish between loss of histones versus altered modifications

  • For accurate quantification, normalize histone H4 signals to multiple housekeeping proteins rather than a single reference

  • Consider the effect of post-translational modifications on antibody recognition, particularly in aging studies where modification patterns change

How does HIST1H4A (Ab-59) Antibody compare with other histone H4 antibodies that target different modifications?

The antibody landscape for histone H4 research includes numerous reagents targeting various modifications and epitopes. Understanding these differences is crucial for experimental design:

When selecting between these antibodies, researchers should consider:

• The specific biological question being addressed (general H4 dynamics vs. specific modifications)

• The technique being employed (some modifications are better detected by certain methods)

• Potential cross-reactivity between modifications (validation with peptide arrays is recommended)

What methods should be used to validate HIST1H4A (Ab-59) Antibody specificity in epigenetic studies?

Rigorous validation of antibody specificity is essential in histone research to avoid misleading results. The following validation approach is recommended:

• Peptide competition assay:

  • Pre-incubate antibody with immunizing peptide before application

  • Should abolish specific signal if antibody is specific

• MODified™ Histone Peptide Array testing:

  • Allows screening against 384 histone tail peptides with 59 different modifications

  • Provides comprehensive specificity profile and identifies cross-reactivity

• Knockout/knockdown validation:

  • Use of HIST1H4A knockdown models or systems with conditional histone depletion

  • Comparison with other histone H4 gene family members to confirm specificity

• Mass spectrometry correlation:

  • Confirmation of antibody-detected modifications by mass spectrometry

  • Essential for quantitative studies of histone modifications

• Cross-platform validation:

  • Test antibody in multiple applications (e.g., WB, ChIP, IF) to ensure consistent reactivity

  • Different applications may reveal distinct specificity profiles

Research shows that up to 25% of commercially available histone antibodies may exhibit significant cross-reactivity or fail validation tests, highlighting the importance of thorough validation .

How can HIST1H4A (Ab-59) Antibody be used to investigate the genomic distribution of histone H4?

The genomic distribution of histone H4 and its modifications can be systematically mapped using HIST1H4A (Ab-59) Antibody through several approaches:

• ChIP-seq experimental design:

  • Input normalization is critical due to the widespread distribution of histone H4

  • Spike-in controls with chromatin from another species can improve quantification accuracy

  • Sequential ChIP (Re-ChIP) can reveal co-occurrence of different modifications

• Analysis considerations:

  • Focus on relative enrichment patterns rather than binary presence/absence

  • Compare with datasets for specific modifications (H4K5ac, H4K12ac, etc.) to identify functional domains

  • Account for nucleosome occupancy changes in different cellular states

• Biological insights:

  • Studies in aging models show region-specific loss of histone H4, with telomeric regions often showing earlier depletion

  • Cancer models reveal altered distribution patterns of histone H4, particularly around oncogenes

  • Nucleosome occupancy changes measured with this antibody can provide insights into chromatin accessibility changes

What are the critical considerations when designing multiplexed immunoassays with HIST1H4A (Ab-59) Antibody?

Multiplexed detection of histone modifications presents unique challenges and opportunities:

• Antibody compatibility assessment:

  • Test for cross-reactivity between antibodies in the multiplexed panel

  • Select antibodies raised in different host species to enable differentiation

  • Consider using directly conjugated primary antibodies to avoid secondary antibody cross-reactivity

• Sequential staining protocols:

  • For immunofluorescence or immunohistochemistry applications, optimize order of antibody application

  • Include thorough blocking steps between applications

  • Consider spectral overlap when selecting fluorophores for multiplexed detection

• Quantitative considerations:

  • Establish individual staining controls for each antibody

  • Perform quantification against standard curves for each target

  • Account for potential epitope masking when multiple antibodies target the same protein

• Technical validation:

  • Flow cytometry can validate co-localization quantitatively at single-cell resolution

  • Mass cytometry (CyTOF) offers high-dimensional analysis of multiple histone modifications

  • Imaging mass cytometry enables spatial resolution of multiple modifications in tissue context

How does nucleosome occupancy impact the interpretation of HIST1H4A (Ab-59) Antibody results?

Understanding nucleosome dynamics is essential for correctly interpreting histone H4 antibody signals:

• Experimental challenges:

  • Reduced antibody signal can reflect either histone loss or epitope masking due to modifications

  • Nucleosome occupancy changes can occur independently of histone protein level changes

  • Chromatin accessibility affects antibody penetration in fixed samples

• Biological context:

  • Age-related research shows reduced nucleosome occupancy with aging in human skin fibroblasts

  • Yeast models demonstrate 50-75% reduction in histone occupancy in aged populations

  • Cancer cells show altered nucleosome positioning around key regulatory elements

• Integrated approaches:

  • Combine ChIP-seq with ATAC-seq or DNase-seq to correlate histone signals with chromatin accessibility

  • Use nascent RNA analysis (e.g., GRO-seq) to correlate histone signals with transcriptional activity

  • Employ micrococcal nuclease (MNase) digestion patterns to map nucleosome positions in relation to histone signals

Recent studies emphasize that histone depletion in aging does not affect nucleosome spacing uniformly but changes nucleosome occupancy with loss typically concentrated in nucleosome-poor regions of the chromatin .

What role do histone variants play in the interpretation of HIST1H4A (Ab-59) Antibody results?

The existence of histone variants necessitates careful consideration when interpreting antibody signals:

• Genomic complexity of H4 genes:

  • Humans have multiple genes encoding identical or nearly identical H4 proteins

  • HIST1H4A is located in the HIST1 cluster on chromosome 6, containing approximately a55 histone genes

  • Additional H4 genes are found in HIST2 and HIST3 clusters on chromosome 1, and HIST4 locus on chromosome 12

• Experimental considerations:

  • The HIST1H4A (Ab-59) Antibody may detect products from multiple H4 gene loci that share sequence homology

  • Quantitative PCR targeting specific H4 transcripts can complement protein-level antibody studies

  • Consider potential differential regulation of H4 variants in different cell types or physiological states

• Research applications:

  • In cancer research, aberrant expression of specific histone variants has been linked to more aggressive phenotypes

  • Evolutionary conservation of H4 makes this antibody potentially useful across species, though validation is required

  • Target epitope conservation should be confirmed when studying model organisms