HIST1H4A (Ab-5) Antibody

Basic Research Questions

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

  HIST1H4A (Ab-5) Antibody is a polyclonal antibody that specifically recognizes histone H4 when acetylated at lysine 5 (Lys5). This antibody is designed to detect endogenous levels of acetylated histone H4 at this specific position without cross-reacting with non-acetylated histone H4 . The antibody recognizes a peptide sequence surrounding the Lys5 site derived from human histone H4 . This post-translational modification is associated with transcriptional activation and various DNA repair processes, making it a valuable target for epigenetic research .

• What are the validated experimental applications for HIST1H4A (Ab-5) Antibody?

  The HIST1H4A (Ab-5) Antibody has been validated for multiple experimental techniques with specific dilution recommendations:

  Application	Recommended Dilution	Notes
  Western Blotting (WB)	1:500-5000	Detects a specific band at ~12 kDa
  Immunohistochemistry (IHC-P)	1:1-100	For paraffin-embedded tissues
  Immunofluorescence (IF)	1:50-200	Nuclear localization expected
  Immunoprecipitation (IP)	1:200-2000	For protein complex isolation
  Chromatin Immunoprecipitation (ChIP)	1:25	For DNA-protein interaction studies
  ELISA	As recommended	For quantitative detection

  For optimal ChIP results, it's recommended to use 20 μl of antibody and 10 μg of chromatin (approximately 4 × 10^6 cells) per immunoprecipitation .

• What is the species reactivity profile of HIST1H4A (Ab-5) Antibody?

  According to the product information, the HIST1H4A (Ab-5) Antibody has confirmed reactivity with both human and mouse samples . This makes it suitable for comparative studies across these mammalian species. The antibody targets a highly conserved region of histone H4, which explains its cross-species reactivity. The high degree of conservation is evident from the search results showing that "human histone 4 is identical in aa sequence to mouse histone 4" . When designing experiments, it's important to note that while the antibody may potentially react with other species based on sequence homology, such reactivity should be experimentally validated before use in research applications.

• What are the optimal storage conditions for maintaining HIST1H4A (Ab-5) Antibody activity?

  To maintain optimal antibody performance, HIST1H4A (Ab-5) Antibody should be:

  • Refrigerated at 2-8°C for short-term storage (up to 2 weeks)

  • Stored at -20°C in small aliquots for long-term preservation

  • Protected from repeated freeze-thaw cycles which can degrade antibody activity

  • Maintained in its buffer solution containing 0.03% Proclin 300 and 50% Glycerol

  The expiration date is typically 12 months from the date of receipt when properly stored . For research requiring consistent antibody performance across multiple experiments, creating single-use aliquots upon receipt is highly recommended.

Advanced Research Questions

• How can researchers optimize ChIP-seq protocols with HIST1H4A (Ab-5) Antibody to accurately map genome-wide H4K5ac distribution?

  Optimizing ChIP-seq with HIST1H4A (Ab-5) Antibody requires careful attention to multiple experimental parameters:

  • Chromatin preparation: Use 10 μg of chromatin (approximately 4 × 10^6 cells) per immunoprecipitation

  • Antibody amount: 20 μl of antibody per reaction for optimal enrichment

  • Crosslinking optimization: Typical formaldehyde crosslinking for histone modifications is 10-15 minutes, but this may require optimization

  • Sonication calibration: Aim for consistent chromatin fragmentation of 200-500 bp

  • Washing stringency: Balance between signal retention and background reduction

  • Controls: Include input samples, IgG controls, and known positive regions

  It's advisable to validate the antibody using known H4K5ac-enriched genomic regions. Research suggests H4K5ac is often associated with active transcription start sites and enhancer regions. The SimpleChIP® Enzymatic Chromatin IP Kits have been validated for use with this antibody . When analyzing data, compare H4K5ac distribution with other histone modifications like H4K12ac, which shows enrichment on lagging strands during DNA replication .

• What methodological approaches can resolve contradictory results when studying H4K5ac in differentiation models?

  Contradictory results in differentiation studies can arise from various factors. To resolve these inconsistencies:

  • Standardize cell synchronization: Cell cycle variations significantly affect histone acetylation patterns

  • Consider tissue specificity: Different tissues show distinct H4K5ac patterns during development

  • Temporal resolution: Collect samples at multiple closely-spaced timepoints to capture transient modifications

  • Control for cellular heterogeneity: Use cell sorting or single-cell approaches when possible

  • Integrate multiple techniques: Combine ChIP-seq, immunofluorescence, and Western blotting data

  The search results indicate that histone H4 gene expression varies significantly during development. For example, "Hist1h4a, Hist1h4d, and Hist4h4 genes showed the same pattern across retinal development" with expression dipping at E18 followed by a spike at P0 and then a steady decline during postnatal development . These temporal dynamics must be considered when interpreting seemingly contradictory results from different developmental timepoints.

• How can researchers distinguish between specific H4K5ac signal and cross-reactivity with other acetylated lysine residues on histone H4?

  Distinguishing specific H4K5ac signal from cross-reactivity requires rigorous controls:

  • Peptide competition assays: Compare antibody binding in the presence of H4K5ac peptides versus other acetylated H4 peptides

  • Knockout/knockdown controls: Use cells with reduced H4K5ac (via HAT inhibitors or genetic approaches) as negative controls

  • Mutant histones: When possible, use cell lines expressing H4K5R mutants (which cannot be acetylated) as controls

  • Multiple antibodies: Compare signals from different H4K5ac antibodies with distinct epitopes

  • Correlation with HAT activity: Confirm that conditions altering known H4K5 acetyltransferases (like Esa1p, Tip60, or CBP/p300 ) correspondingly affect your signal

  Western blot validation should show a single specific band at approximately 12 kDa under reducing conditions . If multiple bands appear, further validation is necessary before proceeding with more complex applications.

• What are the optimal experimental designs for investigating the role of H4K5ac in DNA damage response pathways?

  To effectively study H4K5ac in DNA damage response:

  • Time-course experiments: Collect samples at multiple timepoints (5min, 15min, 30min, 1h, 4h, 24h) after damage induction

  • Damage-specific induction: Compare H4K5ac patterns after different types of DNA damage (UV, ionizing radiation, replication stress)

  • Co-localization studies: Examine spatial relationships between H4K5ac and DNA damage markers (γH2AX, 53BP1)

  • Pathway inhibition: Use inhibitors of specific DNA repair pathways to determine which processes involve H4K5ac

  • HAT/HDAC manipulation: Alter the activities of enzymes known to modify H4K5 and assess repair efficiency

  The search results indicate that "H4K5 acetylation by Esa1p in yeast or Tip60 in mammalian cells may contribute to both transcriptional activation and DNA repair, including non-homologous end joining and replication-coupled repair" . This provides a foundation for investigating which specific repair pathways involve H4K5ac and how the timing of acetylation correlates with repair progression.

• How can HIST1H4A (Ab-5) Antibody be used in combination with other histone modification antibodies to study the histone code?

  Multiparameter analysis of histone modifications requires careful experimental design:

  • Sequential ChIP (re-ChIP): Perform first immunoprecipitation with HIST1H4A (Ab-5) Antibody, then re-immunoprecipitate with antibodies against other modifications

  • Multiplex immunofluorescence: Use directly conjugated antibodies with distinct fluorophores to visualize multiple modifications simultaneously

  • Correlated Western blotting: Run parallel blots with the same samples to compare modification patterns

  • ChIP-seq integration: Analyze overlapping and distinct genomic regions enriched for different modifications

  • Normalization strategy: Always normalize modification-specific signals to total histone H4 levels

  When designing these experiments, consider that H4K5ac often co-occurs with other acetylation marks like H4K12ac . Research suggests distinct patterns of histone modifications during DNA replication, with H4K20me2 showing strong leading strand bias and H4K12ac showing lagging strand bias . Understanding these relationships can provide insights into the functional consequences of combinatorial histone modifications.

• What are the technical considerations for using HIST1H4A (Ab-5) Antibody to study the inheritance of histone modifications during DNA replication?

  Studying histone modification inheritance during replication requires specialized approaches:

  • Cell synchronization: Use methods like double thymidine block or nocodazole treatment to obtain populations enriched in specific cell cycle phases

  • Pulse-chase experiments: Label newly synthesized histones and track their modification status over time

  • Nascent chromatin capture: Isolate newly replicated DNA to study associated histone modifications

  • Single-molecule imaging: Use super-resolution microscopy to visualize H4K5ac dynamics during replication

  • Replication factor co-IP: Examine interactions between H4K5ac and components of the replication machinery

  The search results indicate that "DNA polymerase α (Pol α), which synthesizes short primers for DNA synthesis, binds histone H3-H4 preferentially" and that mutation of its histone-binding motif "impairs parental histone transfer to lagging strand" . This suggests a potential role for replication machinery in histone deposition and modification maintenance, which could be studied using the HIST1H4A (Ab-5) Antibody.

• How should researchers interpret variations in H4K5ac levels across different cell types and disease states?

  Interpreting H4K5ac variations requires contextual analysis:

  • Baseline establishment: Determine normal H4K5ac levels in relevant healthy cell types

  • Normalization approach: Always normalize to total H4 levels to account for differences in histone abundance

  • Multi-omic integration: Correlate H4K5ac changes with transcriptome, proteome, and other epigenetic marks

  • Cell-type specific markers: Use cell identity markers to ensure comparisons between equivalent cell populations

  • Developmental timing: Consider that histone acetylation patterns change during development and differentiation

  Research on histone H4 expression shows that levels can vary significantly during development. For instance, "the expression of Hist1h4a, Hist1h4d, and Hist4h4 genes... dipped at E18 followed by a spike at P0 and then a steady decline during postnatal development" . These natural variations must be considered when comparing H4K5ac levels between different conditions or disease states.

• What methodological approaches can identify proteins that interact with H4K5ac in chromatin regulation?

  To identify H4K5ac-interacting proteins:

  • Acetyl-lysine reader domain screening: Test interaction of known acetyl-lysine readers with H4K5ac peptides

  • SILAC-based proteomic approaches: Compare proteins binding to acetylated versus non-acetylated H4K5 peptides

  • Proximity labeling: Use BioID or APEX2 fused to H4 to identify proteins in close proximity to H4K5ac sites

  • IP-mass spectrometry: Use HIST1H4A (Ab-5) Antibody to immunoprecipitate H4K5ac and associated proteins

  • Cross-linking approaches: Apply formaldehyde or DSS cross-linking to capture transient interactions

  The search results mention that "H4K5 is acetylated by multiple HAT proteins including Esa1p, Tip60, and CBP/p300" . These known interactors provide a starting point for investigating the broader network of proteins that recognize or are influenced by H4K5 acetylation.

• How can researchers effectively use HIST1H4A (Ab-5) Antibody to investigate the relationship between H4K5ac and RNA processing?

  To explore connections between H4K5ac and RNA processing:

  • ChIP-seq with RNA-seq integration: Correlate H4K5ac enrichment with alternative splicing patterns

  • Nuclear fractionation: Compare H4K5ac levels in chromatin associated with active transcription/splicing

  • Co-IP with splicing factors: Test interactions between H4K5ac and components of the spliceosome

  • Transcription elongation rate analysis: Investigate whether H4K5ac affects RNA polymerase II processivity

  • Manipulation of H4K5ac levels: Observe effects on splicing after altering H4K5 acetylation status

  While the direct role of H4K5ac in RNA processing isn't explicitly mentioned in the search results, related histone proteins show interesting connections to splicing. For example, "H1.5 is enriched around splicing sites, especially on genes that are highly alternatively spliced" and "H1.5 occupancy causes RNA polymerase II pausing" . Given the interplay between different histone modifications in regulating chromatin structure and gene expression, investigating potential roles of H4K5ac in RNA processing could yield important insights.

• What are the current limitations of using HIST1H4A (Ab-5) Antibody in single-cell epigenomic studies?

  Single-cell applications face several technical challenges:

  • Sensitivity limitations: Standard ChIP protocols require thousands to millions of cells

  • Antibody specificity: Background binding becomes more problematic at the single-cell level

  • Epitope accessibility: Fixation conditions critical for maintaining single-cell integrity may affect antibody binding

  • Signal amplification: Methods needed to detect H4K5ac in individual cells without introducing bias

  • Validation approaches: Limited material makes orthogonal validation difficult

  Emerging techniques like CUT&Tag, CUT&RUN, or single-cell ChIP-seq adaptations may overcome some of these limitations. These methods can reduce input requirements and improve signal-to-noise ratios. Researchers should consider pilot studies comparing bulk and single-cell results using the same antibody lot to understand potential biases or limitations before proceeding with large-scale single-cell studies.