HIST1H4A (Ab-91) Antibody

What is HIST1H4A (Ab-91) Antibody and what epitope does it specifically recognize?

HIST1H4A (Ab-91) Antibody is a polyclonal antibody raised in rabbits against a peptide sequence surrounding lysine 91 (K91) in human Histone H4. This antibody specifically recognizes the region containing this lysine residue, which is located not in the commonly studied N-terminal tail but within the globular core domain of histone H4 .

The antibody's specificity has been validated through dot blot analysis against modified and unmodified peptides encompassing several sites of H4 acetylation, demonstrating high specificity for acetylated H4K91 . Importantly, this antibody recognizes histone H4 isolated from mammalian cells (e.g., HeLa) but fails to recognize recombinant H4 produced in E. coli, which lacks post-translational modifications .

What experimental applications has the HIST1H4A (Ab-91) Antibody been validated for?

The HIST1H4A (Ab-91) Antibody has been validated for multiple research applications:

• Enzyme-Linked Immunosorbent Assay (ELISA)

• Immunohistochemistry (IHC), including paraffin-embedded tissues

• Immunofluorescence (IF)

• Western Blotting (WB)

• Chromatin Immunoprecipitation (ChIP)

For optimal experimental conditions, the following dilutions are recommended:

• IHC-P: 1:10-1:200 (starting with 1:20 for optimization)

• IF: 1:50-1:200

• ELISA: Follow manufacturer's protocol

What is the biological significance of histone H4 lysine 91 in chromatin structure?

Lysine 91 in histone H4 occupies a critical position at the interface between histone H3/H4 tetramers and H2A/H2B dimers, making it structurally significant for nucleosome stability . This strategic location means that modifications at this residue can directly influence:

• Stability of the histone octamer

• Higher-order chromatin structure

• Nucleosome assembly and disassembly

• Chromatin accessibility for DNA-templated processes

Mutation studies (H4K91A) have demonstrated that altering this residue confers phenotypes consistent with defects in chromatin assembly, including sensitivity to DNA damaging agents and destabilization of the histone octamer . This positions H4K91 as a critical regulatory site for chromatin structure and function.

How should I optimize immunohistochemistry protocols using HIST1H4A (Ab-91) Antibody?

For optimal IHC results with HIST1H4A (Ab-91) Antibody, follow this validated protocol:

• Tissue preparation: After dewaxing and hydration of paraffin-embedded sections, perform antigen retrieval using high pressure in citrate buffer (pH 6.0) .

• Blocking: Block sections with 10% normal goat serum for 30 minutes at room temperature .

• Primary antibody incubation: Dilute the antibody in 1% BSA (starting at 1:20 dilution) and incubate overnight at 4°C .

• Detection system: Use a biotinylated secondary antibody followed by visualization with an HRP-conjugated SP system .

• Controls: Include positive controls (human colon cancer tissue has been validated) and negative controls (omitting primary antibody) .

The antibody has been successfully used on automated systems such as the Leica Bond™ system for standardized results .

What are the best practices for using HIST1H4A (Ab-91) Antibody in ChIP experiments?

When employing HIST1H4A (Ab-91) Antibody for ChIP studies, consider these critical methodological steps:

• Antibody specificity verification: As an additional measure to ensure specificity, preincubate the antibody with lysate isolated from strains containing the H4K91A allele to block any potential cross-reactivity against other histone modifications .

• Controls: Include:

  • Input samples (pre-immunoprecipitation)

  • IgG controls for nonspecific binding

  • H4K91A mutant samples as negative controls

  • Positive control regions (active genes)

  • Negative control regions (telomeres, HMR locus)

• Quantification: Use quantitative PCR to determine the relative abundance of H4K91 acetylation at different genomic loci, comparing enrichment at active genes versus silenced regions .

• Data interpretation: Remember that H4K91 acetylation is significantly enriched in active regions of the genome and present at low levels at telomeres and the HMR locus .

How can I distinguish between acetylation and monoubiquitylation at H4K91 in my experiments?

Distinguishing between different modifications at H4K91 requires careful experimental design:

• Modification-specific antibodies: HIST1H4A (Ab-91) Antibody specifically recognizes the region around H4K91, while modification-specific antibodies are required to distinguish acetylation from monoubiquitylation .

• Site-directed mutagenesis approach: Perform site-directed mutagenesis of histone H4K91 (K→A) to eliminate the possibility of both modifications. Research has shown that this mutation specifically eliminates BBAP-mediated monoubiquitylation of histone H4 .

• Kinetic studies: Track temporal changes in both modifications following stimuli like DNA damage. Research indicates that H4K91ac levels remain stable while monoubiquitylation increases after DNA damage .

• Depletion studies: When BBAP (the E3 ligase responsible for H4K91 monoubiquitylation) is depleted, an increase in acetylated H4K91 is observed, suggesting potential competition between these modifications .

What is the role of H4K91 modification in DNA damage response pathways?

H4K91 modifications play critical roles in DNA damage response:

• Mutation sensitivity: Mutations at H4K91 (K91A) confer phenotypes consistent with defects in DNA repair, including increased sensitivity to DNA damaging agents like MMS .

• Monoubiquitylation signaling: BBAP-mediated monoubiquitylation of H4K91 is induced following DNA damage (e.g., doxorubicin treatment) .

• Histone modification cascade: BBAP-mediated H4K91 monoubiquitylation promotes H4K20 mono- and dimethylation, which are necessary for 53BP1 recruitment to DNA damage sites .

• Pathway integration: H4K91 modification appears to work in concert with other DNA damage response pathways, as genetic studies show increased sensitivity when H4K91 mutations are combined with mutations in certain DNA repair factors .

This indicates H4K91 modification serves as a signaling platform in the DNA damage response cascade, linking chromatin structure to DNA repair protein recruitment.

How does H4K91 modification influence gene silencing and chromatin structure?

H4K91 plays a complex role in gene silencing and chromatin organization:

• Distribution pattern: H4K91 acetylation is enriched in active genomic regions and present at low levels at silenced loci like telomeres and the HMR locus .

• Effects of mutation: Mutation of H4K91 (K91A) causes:

  • Dramatic loss of Sir2 protein from telomeres and HMR

  • Increased H4 N-terminal tail acetylation at silenced regions

  • Significant increase in H3K79 methylation at telomeres

  • Derepression of silenced genes

  • Acquisition of euchromatin-like features at previously silenced regions

• Structural implications: The interface location of H4K91 between H3/H4 tetramers and H2A/H2B dimers suggests its modification affects nucleosome stability and higher-order chromatin structure .

This positions H4K91 modification as a critical regulator of the transition between transcriptionally active and silent chromatin states.

What is the relationship between H4K91 modifications and other histone marks?

Research using HIST1H4A (Ab-91) Antibody and other approaches has revealed interactions between H4K91 modifications and other histone marks:

• Competing modifications: H4K91 acetylation and monoubiquitylation appear to be mutually exclusive or competing modifications at the same residue .

• Cross-talk with H4K20: BBAP-mediated monoubiquitylation of H4K91 promotes H4K20 mono- and dimethylation, necessary for 53BP1 recruitment .

• Influence on H4 N-terminal tail: Mutation of H4K91 leads to increased acetylation of the H4 N-terminal tail at silenced genomic regions, suggesting cross-talk between these modifications .

• Relationship with H3K79 methylation: H4K91 mutation results in increased H3K79 methylation at telomeres, indicating a functional relationship between these modifications in maintaining silent chromatin .

These interactions position H4K91 modification as a central hub in the histone modification network that influences chromatin structure, gene expression, and DNA repair.

What are common challenges when using HIST1H4A (Ab-91) Antibody and how can they be addressed?

When working with HIST1H4A (Ab-91) Antibody, researchers may encounter several technical challenges:

• Cross-reactivity concerns: The antibody should be validated for specificity using:

  • Dot blot analysis against modified and unmodified peptides

  • Comparison of reactivity between mammalian histones and recombinant E. coli-produced H4

  • Pre-incubation with lysate from H4K91A mutant cells to block potential cross-reactivity

• Storage and stability issues: To maintain antibody functionality:

  • Store at -20°C or -80°C for long-term storage

  • Avoid repeated freeze-thaw cycles

  • The antibody is provided in a buffer containing 50% glycerol, 0.01M PBS, pH 7.4, and 0.03% Proclin 300 as a preservative

• Signal optimization in IHC: For optimal immunohistochemical signal:

  • Use high-pressure citrate buffer (pH 6.0) for antigen retrieval

  • Optimize antibody dilution starting from 1:20

  • Incubate with primary antibody overnight at 4°C

How can I confirm the specificity of my H4K91 modification detection experiments?

To verify the specificity of experiments involving H4K91 modifications:

What are potential applications of HIST1H4A (Ab-91) Antibody in cancer research?

Given the role of H4K91 modifications in DNA repair and gene silencing, several applications in cancer research are promising:

• Biomarker potential: Assess H4K91 modification patterns in:

  • Different cancer types and stages

  • Response to DNA-damaging chemotherapeutics

  • Correlation with known DNA repair deficiencies (e.g., BRCA mutations)

• Therapeutic response monitoring: Evaluate changes in H4K91 modifications following:

  • Treatment with HDAC inhibitors

  • DNA-damaging agents like doxorubicin

  • PARP inhibitors

  • Epigenetic modulators

• Mechanistic studies: Investigate how altered H4K91 modification contributes to:

  • Genomic instability in cancer

  • Altered gene expression patterns

  • Chromatin structure abnormalities

  • Resistance to DNA-damaging therapies

How might H4K91 modifications contribute to epigenetic inheritance and chromatin dynamics?

The strategic location of H4K91 at the interface between histone dimers and tetramers suggests several intriguing research directions:

• Chromatin assembly dynamics: Investigate how H4K91 modifications influence:

  • Nucleosome assembly during DNA replication

  • Histone exchange in transcriptionally active regions

  • Maintenance of silent chromatin through cell divisions

• Epigenetic inheritance mechanisms: Explore whether H4K91 modifications:

  • Are preserved during DNA replication and mitosis

  • Contribute to epigenetic memory of transcriptional states

  • Interact with DNA methylation patterns

• Higher-order chromatin structure: Examine how H4K91 modifications affect:

  • Chromatin compaction and decompaction

  • Enhancer-promoter interactions

  • Topologically associating domain (TAD) boundaries

This antibody provides a valuable tool for investigating these fundamental aspects of chromatin biology and epigenetic regulation.