Formyl-HIST1H4A (K77) Antibody

What is HIST1H4A and what is the significance of lysine 77 formylation?

HIST1H4A (Histone H4) is a core component of nucleosomes, essential for chromatin structure and gene regulation. As one of the most highly conserved proteins, histone H4 plays a central role in transcription regulation, DNA repair, DNA replication, and chromosomal stability . Lysine 77 (K77) formylation represents an important post-translational modification (PTM) that contributes to the histone code, potentially affecting chromatin accessibility and gene expression patterns . Unlike better-studied modifications such as acetylation, formylation is an emerging area of epigenetic research with distinct regulatory implications. Formylation at K77 may serve as a unique epigenetic mark that influences nucleosome stability and recruitment of specific regulatory proteins to chromatin regions.

What applications has the Formyl-HIST1H4A (K77) antibody been validated for?

The Formyl-HIST1H4A (K77) Polyclonal Antibody has been specifically validated for the following applications:

This rabbit polyclonal antibody has been specifically validated against human (Homo sapiens) samples and recognizes the peptide sequence surrounding the formylated lysine 77 site derived from Human Histone H4 . The antibody enables researchers to detect and quantify this specific modification in various experimental contexts.

How should I store and handle the Formyl-HIST1H4A (K77) antibody?

For optimal performance and longevity of the Formyl-HIST1H4A (K77) antibody:

• Short-term storage (up to 2 weeks): Maintain refrigerated at 2-8°C

• Long-term storage: Store at -20°C in small aliquots to prevent freeze-thaw cycles

• Buffer composition: Typically preserved in 50% glycerol with 0.03% Proclin 300 as a preservative

• Expiration: Most preparations have a recommended use period of 12 months from receipt when stored properly

• Avoid repeated freeze-thaw cycles as these can degrade antibody performance and specificity

How can I distinguish between formylation and acetylation at K77 of Histone H4?

Distinguishing between formylation and acetylation at K77 requires careful experimental design:

To definitively differentiate these modifications:

• Perform parallel immunoblots with both Formyl-K77 and Acetyl-K77 specific antibodies

• Include appropriate controls (e.g., unmodified peptides)

• Consider mass spectrometry analysis for unambiguous identification

• Compare elution profiles and fragmentation patterns of modified peptides

The specific Formyl-HIST1H4A (K77) antibody recognizes the formyl group at K77 which has a distinct molecular structure compared to the acetyl group recognized by Histone H4 (acetyl K77) antibody . These antibodies have been specifically designed to distinguish between these closely related but functionally distinct modifications.

What is the relationship between K77 formylation and other histone modifications?

The relationship between K77 formylation and other modifications reflects the complex interplay within the histone code:

• Modification Crosstalk: Formylation at K77 may influence or be influenced by adjacent modifications such as methylation or acetylation at nearby residues including K79

• Combinatorial Effects: The specific combination of K77 formylation with other modifications creates distinct "epigenetic signatures" that may recruit different regulatory proteins

• Temporal Dynamics: Formylation at K77 may precede or follow other modifications in response to cellular signals, establishing a temporal hierarchy of histone modifications

• Functional Competition: Formylation at K77 may compete with other modifications at the same residue (e.g., acetylation, methylation) with potential differential effects on chromatin structure

Advanced ChIP-seq experiments combined with other PTM-specific antibodies can help elucidate these relationships by mapping the co-occurrence patterns of formylation and other modifications across the genome.

What is the optimal protocol for ChIP experiments using Formyl-HIST1H4A (K77) antibody?

For optimal ChIP experiments with Formyl-HIST1H4A (K77) antibody:

• Crosslinking and Sonication:

  • Crosslink cells with 1% formaldehyde for 10 minutes at room temperature

  • Quench with 125mM glycine for 5 minutes

  • Lyse cells and sonicate chromatin to 200-500bp fragments

• Immunoprecipitation:

  • Pre-clear chromatin with protein A/G beads

  • Incubate cleared chromatin with 2-5μg Formyl-HIST1H4A (K77) antibody overnight at 4°C

  • Add protein A/G beads and incubate for 2-3 hours

  • Perform stringent washes to remove non-specific binding

• Controls:

  • Input control (non-immunoprecipitated chromatin)

  • IgG control (non-specific antibody of same isotype)

  • Positive control regions (known formylated regions)

  • Negative control regions (regions typically lacking formylation)

• Analysis Methods:

  • qPCR for targeted analysis of specific genomic regions

  • ChIP-seq for genome-wide profiling of K77 formylation distribution

ChIP experiments have been successfully validated using this antibody, allowing for specific enrichment of genomic regions associated with formylated H4K77 .

How should I optimize Western blotting conditions for detecting Formyl-HIST1H4A (K77)?

For optimal Western blot results with Formyl-HIST1H4A (K77) antibody:

Critical steps for successful detection:

• Include deformylase inhibitors in extraction buffers to prevent loss of formylation

• Always use fresh samples as formyl groups can be unstable

• Include positive controls (cell lines known to exhibit K77 formylation)

• Compare with other histone H4 antibodies to confirm band identity

Western blot analysis has been successfully performed on various cell lysates including HeLa, HepG2, and MCF-7 cells using this antibody, showing specific bands at approximately 11 kDa corresponding to histone H4 .

What are common issues in Formyl-HIST1H4A (K77) antibody experiments and how can I address them?

When analyzing data from Formyl-HIST1H4A (K77) antibody experiments, consider:

• Always normalize to appropriate controls (input for ChIP, loading controls for Western blot)

• Compare results across multiple experimental methods when possible

• Consider the biological context and cell type-specific patterns of formylation

• Be aware that formylation levels may change dramatically under different cellular conditions

How can I validate the specificity of the Formyl-HIST1H4A (K77) antibody?

To confirm antibody specificity for formylated K77:

• Peptide Competition Assays:

  • Pre-incubate antibody with formylated K77 peptide versus unmodified peptide

  • If signal disappears only with formylated peptide, this confirms specificity

• Knockdown/Knockout Controls:

  • Use genetic approaches to reduce histone H4 expression

  • Verify diminished signal correlates with reduced target protein

• Mass Spectrometry Validation:

  • Perform immunoprecipitation followed by mass spectrometry

  • Confirm enrichment of formylated K77 peptides

• Comparison with Other PTM-Specific Antibodies:

  • Test parallel samples with antibodies against acetyl-K77

  • Different patterns confirm distinction between modifications

• Dot Blot Analysis:

  • Test antibody against a panel of modified and unmodified peptides

  • Quantify binding affinity and cross-reactivity

The manufacturer has validated this antibody for specificity through multiple applications including ELISA, Western blot, and ChIP, confirming its selective recognition of the formylated K77 epitope on histone H4 .

What is the current understanding of the biological significance of histone H4K77 formylation?

Current research suggests several important biological roles for H4K77 formylation:

• Gene Regulation: Emerging evidence suggests formylation at H4K77 may influence transcriptional activity by altering chromatin accessibility and recruiting specific regulatory proteins to marked regions

• Cellular Metabolism: Histone formylation appears linked to cellular metabolic state, potentially serving as a mechanism by which metabolic changes can influence gene expression patterns

• Stress Response: Preliminary data indicates increased H4K77 formylation occurs during certain cellular stress conditions, suggesting a role in stress-responsive gene regulation

• Disease Relevance: Alterations in histone formylation patterns may contribute to pathological states, though this area remains under investigation

• Environmental Response: H4K77 formylation may represent a mechanism for epigenetic response to environmental factors

While research specifically on K77 formylation is still emerging, histone formylation generally represents an exciting frontier in epigenetic research, potentially providing new insights into chromatin regulation mechanisms and disease processes .

What emerging techniques might enhance the study of histone H4K77 formylation?

Several cutting-edge approaches promise to advance H4K77 formylation research:

Integration of these advanced techniques with traditional antibody-based methods will likely provide deeper insights into the functional significance and regulatory mechanisms of H4K77 formylation in diverse biological contexts. As more researchers adopt these approaches, our understanding of this modification's role in the histone code will continue to expand .

How does formylation at K77 compare with formylation at K79 on histone H4?

A comparative analysis reveals important distinctions between these neighboring formylation sites:

Research comparing these two formylation sites is still evolving, but preliminary data suggests they may serve distinct biological functions. The availability of site-specific antibodies for both K77 and K79 formylation enables comparative studies that may reveal important insights into the functional significance of these closely positioned modifications .

Their proximity on the histone tail raises interesting questions about potential interactions or competitive relationships between these modifications, which future research using these specific antibodies can help address.

What are the optimal protocols for immunofluorescence detection of Formyl-HIST1H4A (K77)?

For successful immunofluorescence detection of formylated H4K77:

• Cell Preparation:

  • Fix cells with 4% paraformaldehyde for 15 minutes at room temperature

  • Permeabilize with 0.2% Triton X-100 for 10 minutes

  • Block with 5% BSA in PBS for 1 hour

• Antibody Incubation:

  • Dilute Formyl-HIST1H4A (K77) antibody at 1:50-1:200

  • Incubate overnight at 4°C in a humidified chamber

  • Use fluorophore-conjugated secondary antibody at manufacturer's recommended dilution

• Critical Controls:

  • Secondary antibody only (to assess background)

  • Peptide competition (pre-incubation with formylated K77 peptide)

  • Parallel staining with other histone H4 antibodies for comparison

• Imaging Considerations:

  • Use confocal microscopy for optimal resolution of nuclear signals

  • Acquire z-stacks to capture the three-dimensional distribution

  • Consider co-staining with markers of different chromatin states (heterochromatin vs. euchromatin)

• Analysis Approaches:

  • Quantify nuclear signal intensity

  • Assess colocalization with other histone marks or nuclear structures

  • Compare formylation patterns across different cell cycle stages