Formyl-HIST1H4A (K31) Antibody

What is the Formyl-HIST1H4A (K31) Antibody and what epitope does it recognize?

The Formyl-HIST1H4A (K31) Antibody is a polyclonal antibody raised in rabbits that specifically recognizes the formylation of lysine 31 on histone H4 proteins. This antibody binds to a peptide sequence surrounding the formylated lysine 31 residue derived from human histone H4 . Histone H4 is a core component of nucleosomes that wrap and compact DNA into chromatin, limiting DNA accessibility to cellular machineries . The H4K31 residue is located at the N-terminus of the H4 α1 helix with its side chain extending into the major groove of DNA, making it a structurally significant position for chromatin regulation .

What are the validated experimental applications for this antibody?

Based on current validation studies, the Formyl-HIST1H4A (K31) Antibody has been confirmed effective for:

• Enzyme-Linked Immunosorbent Assay (ELISA)

• Western Blotting (WB)

These applications make the antibody suitable for both quantitative detection and qualitative visualization of formylated H4K31 in various experimental settings. When using this antibody in Western blotting, researchers have observed a predicted band size of approximately 11 kDa, consistent with histone H4's molecular weight .

How does H4K31 formylation differ from other modifications at this residue?

The lysine 31 position of histone H4 can undergo multiple post-translational modifications, including formylation, acetylation, and methylation. Research indicates that these modifications occur in a mutually exclusive manner and serve distinct functions in chromatin regulation :

The H4K31 residue is particularly significant because it lies at the protein-DNA interface close to the dyad axis of the nucleosome, making modifications at this site potentially impactful for chromatin structure and gene regulation .

What protocols should researchers follow for optimal Western blot results?

For optimal Western blot results with the Formyl-HIST1H4A (K31) Antibody, researchers should consider these methodological recommendations:

• Sample preparation: Extract histones using an acid extraction protocol to enrich for histone proteins

• Gel selection: Use 15-18% SDS-PAGE gels to properly resolve low molecular weight histone proteins

• Transfer conditions: Optimize for small proteins (typically 30V for 90 minutes)

• Blocking: Use 5% BSA in TBST as blocking buffer, as demonstrated in published protocols

• Antibody dilution: 1:500 dilution works effectively for Western blotting applications

• Detection system: Secondary antibody conjugated to peroxidase at 1:1000 dilution has proven effective

• Controls: Include unmodified H4 controls and other H4K31 modification controls to confirm specificity

The expected band size is approximately 11 kDa, consistent with observations in NIH/3T3 mouse embryo fibroblast cells .

How can researchers confirm antibody specificity for formylated H4K31?

Confirming specificity is crucial when working with histone modification antibodies. Researchers should implement these methodological approaches:

• Peptide competition assays: Pre-incubate the antibody with formylated H4K31 peptides to demonstrate signal reduction

• Dot blot assays: Test against a panel of modified and unmodified peptides, similar to the approach used for H4K31ac antibody validation

• Cross-reactivity testing: Verify no cross-reactivity with other histone modifications, particularly acetylation at H4K31

• Knockout/knockdown validation: If possible, use cells with mutations at H4K31 that prevent formylation

• Mass spectrometry correlation: Compare antibody-based detection with mass spectrometry identification of formylated H4K31

Research on related modifications demonstrates that careful validation through dot-blot assays against unmodified peptides and peptides with similar modifications is effective in confirming specificity .

What is the biological significance of H4K31 formylation in chromatin biology?

The formylation of H4K31 represents an important post-translational modification with potential significance for chromatin structure and gene regulation. While research on this specific modification is still emerging, insights can be drawn from studies of other modifications at this site:

• Structural impact: The H4K31 residue is located at the protein-DNA interface near the nucleosome dyad axis. Modifications at this site likely alter DNA-histone interactions, potentially affecting nucleosome stability

• Chromatin accessibility: Similar to acetylation at this position, formylation may neutralize the positive charge of lysine, potentially destabilizing the protein-DNA interface and promoting a more open chromatin state

• Transcriptional regulation: Given that H4K31 acetylation is associated with active promoters and H4K31 methylation with repressed genes, formylation may play a distinct regulatory role in gene expression

• Relationship to cellular metabolism: Formylation of lysine residues has been linked to metabolic processes in cells

Understanding how H4K31 formylation fits within the broader histone code will require further genome-wide mapping and functional studies.

How does H4K31 formylation compare across different species and cell types?

While comprehensive comparative studies of H4K31 formylation across species are still limited, we can make some observations based on available research:

• Conservation: Histone H4 is highly conserved across eukaryotes, with the K31 residue being present in humans, mice, and apicomplexan parasites like T. gondii and P. falciparum

• Cell type distribution: The distribution pattern of H4K31 formylation across different cell types remains to be fully characterized

• Species-specific patterns: In P. falciparum, H4K31 acetylation shows a nuclear punctate pattern throughout the intraerythrocytic developmental cycle, suggesting potential specialized transcription factories

• Evolutionary significance: The conservation of this site across diverse species suggests important functional roles

Researchers should consider these cross-species and cell-type variations when designing experiments and interpreting results involving H4K31 formylation studies.

How do histone deacetylase inhibitors (HDACi) affect H4K31 formylation patterns?

The relationship between histone deacetylase inhibitors and H4K31 formylation merits careful investigation. Research on the related modification H4K31ac provides some insights:

• HDACi effects: Cyclopeptide HDACi compounds significantly enhance H4K31ac levels in parasite nuclei, indicating that this modification is regulated by specific HDACs

• Enzyme specificity: Evidence suggests that TgHDAC3 (in T. gondii) may be involved in regulating H4K31 acetylation, based on point mutation studies that abolish enzyme sensitivity to cyclic tetrapeptide compounds

• Comparative responses: While HDACi treatment increases H4K31ac signals, other histone modifications like H3K14ac and H3K27ac may remain unaltered under the same conditions, indicating specific regulatory pathways

For researchers studying H4K31 formylation, examining how various HDACi treatments affect this modification could provide insights into its regulatory mechanisms and relationship to acetylation at the same residue.

What ChIP-seq optimization strategies are recommended for mapping H4K31 formylation patterns?

When designing ChIP-seq experiments to map H4K31 formylation genome-wide, researchers should consider these methodological optimizations:

• Crosslinking conditions: Optimize formaldehyde concentration and fixation time, as histone-DNA interactions at the nucleosome dyad may require different crosslinking conditions than tail modifications

• Sonication parameters: Adjust sonication to generate fragments of 150-300 bp for optimal resolution of nucleosome-level modifications

• Antibody validation: Thoroughly validate the antibody's specificity in ChIP applications before proceeding to sequencing

• Controls:

  • Input controls to normalize for biases in chromatin preparation

  • IgG controls to account for non-specific binding

  • Spike-in controls for quantitative comparisons between samples

• Analysis pipeline: Implement peak calling algorithms optimized for histone modifications rather than transcription factor binding sites

The mutual exclusivity observed between H4K31 acetylation and methylation suggests that comparative ChIP-seq experiments examining multiple modifications at this residue may yield valuable insights into their regulatory relationships.

What is the relationship between H4K31 formylation and other histone modifications in gene regulation?

Understanding how H4K31 formylation integrates with other histone modifications is crucial for deciphering its role in the histone code:

• Mutually exclusive modifications: Research on H4K31 indicates that acetylation and methylation at this position occur in a mutually exclusive manner, suggesting that formylation may similarly exclude other modifications at this site

• Genomic distribution patterns: H4K31ac is enriched at gene promoters while H4K31me1a is found in gene bodies or pericentromeric heterochromatin (in P. falciparum)

• Functional interplay: The positioning of H4K31 at the nucleosome dyad axis suggests potential interactions with other modifications that regulate chromatin accessibility

• Cross-talk mechanisms: Investigating whether enzymes that write or erase H4K31 formylation are influenced by neighboring modifications would be valuable

Research examining the co-occurrence or mutual exclusivity of H4K31 formylation with modifications on other histones would provide insights into its role in higher-order chromatin regulation.

What are common technical challenges when working with Formyl-HIST1H4A (K31) Antibody?

Researchers working with this antibody should be aware of several potential technical challenges:

• Specificity concerns: Ensuring the antibody does not cross-react with other modifications at K31 (acetylation, methylation) or formylation at other lysine residues

• Sensitivity limitations: Formylation may be present at low abundance, requiring optimized detection methods

• Sample preparation issues: Acid extraction methods for histones may affect some modifications

• Batch-to-batch variability: Polyclonal antibodies can show variation between production lots

• Background signal: Optimizing blocking conditions to reduce non-specific binding

To address these challenges, thorough validation of each antibody lot is recommended, along with inclusion of appropriate positive and negative controls in experiments.

How can researchers integrate H4K31 formylation data with other epigenomic datasets?

For comprehensive epigenomic analysis including H4K31 formylation data, researchers should consider:

• Multi-omics integration approaches:

  • Correlate H4K31 formylation with transcriptome data (RNA-seq)

  • Compare with other histone modifications (ChIP-seq)

  • Integrate with chromatin accessibility data (ATAC-seq, DNase-seq)

  • Correlate with DNA methylation patterns

• Analytical frameworks:

  • Use genome browsers for visualization of multiple datasets

  • Apply machine learning approaches to identify patterns across datasets

  • Implement statistical methods for correlation analysis between different epigenetic marks

• Functional validation:

  • Design experiments to manipulate H4K31 formylation levels and observe effects on other epigenetic marks

  • Use CRISPR-based approaches to mutate H4K31 and assess impacts on chromatin organization

Such integrated approaches will help place H4K31 formylation within the broader context of epigenetic regulation and chromatin biology.

What are promising research avenues for understanding H4K31 formylation in disease contexts?

Several promising research directions could advance our understanding of H4K31 formylation in disease:

• Cancer epigenetics: Investigating whether aberrant H4K31 formylation patterns are associated with cancer progression or therapy response

• Inflammatory conditions: Examining potential links between metabolic changes during inflammation and histone formylation patterns

• Neurodegenerative diseases: Exploring whether age-related changes in histone modifications include alterations in H4K31 formylation

• Infectious diseases: Building on existing research in apicomplexan parasites to understand how H4K31 modifications affect host-pathogen interactions

• Metabolic disorders: Investigating connections between cellular metabolism and histone formylation levels

These research avenues could lead to new insights into disease mechanisms and potentially identify novel therapeutic targets.

What technological innovations might advance H4K31 formylation research?

Emerging technologies that could significantly advance the study of H4K31 formylation include:

• Single-cell epigenomics: Adapting ChIP-seq protocols for single-cell analysis to examine cell-to-cell variation in H4K31 formylation patterns

• Advanced imaging techniques: Developing tools for visualizing H4K31 formylation in living cells to track dynamic changes

• Protein engineering approaches: Creating synthetic readers, writers, or erasers of H4K31 formylation to manipulate this modification in vivo

• Mass spectrometry innovations: Improving sensitivity for detecting and quantifying formylation in complex histone samples

• Computational methods: Developing specialized algorithms for analyzing genome-wide patterns of H4K31 formylation and its relationship to chromatin structure

These technological advances would enable researchers to address fundamental questions about the biological roles and regulatory mechanisms of H4K31 formylation.