HIST1H4A Recombinant Monoclonal Antibody

What is HIST1H4A and what role does it play in chromatin structure?

HIST1H4A is a gene that encodes histone H4, one of the four core histones (H2A, H2B, H3, and H4) that form the nucleosome structure of chromosomal fiber in eukaryotes. Histone H4 is a basic nuclear protein that is essential for chromatin packaging and DNA organization. Two molecules of each core histone form an octamer, around which approximately 146 base pairs of DNA are wrapped in repeating units called nucleosomes . The HIST1H4A gene is intronless and is located within the large histone gene cluster on chromosome 6 .

The protein encoded by HIST1H4A is 103 amino acids in length with a molecular weight of approximately 11,367 daltons . As a component of the nucleosome octamer, histone H4 plays a crucial role in regulating chromatin accessibility and consequently influences gene expression, DNA replication, and DNA repair processes. The post-translational modifications of histone H4, including acetylation and methylation, are key epigenetic marks that contribute to the regulation of these processes.

How are recombinant monoclonal antibodies against HIST1H4A produced?

Recombinant monoclonal antibodies against HIST1H4A are produced using in vitro expression systems rather than traditional hybridoma techniques. The production process involves several key steps:

• Isolation of antibody DNA sequences from immunoreactive rabbits that have mounted a specific immune response against histone H4.

• Cloning of these specific antibody DNA sequences into expression vectors.

• Screening of individual clones to select candidates with optimal binding properties.

• Expression of the antibody in a suitable host system (typically mammalian cells, but also bacteria, yeast, or insect cells).

• Purification and quality control testing of the expressed antibody .

This recombinant production approach offers several advantages over traditional hybridoma-based methods, including:

• Better specificity and sensitivity for the target antigen

• Improved lot-to-lot consistency in antibody performance

• Animal origin-free formulations, reducing potential contaminants

• Broader immunoreactivity due to leveraging the larger rabbit immune repertoire

These characteristics make recombinant rabbit monoclonal antibodies particularly valuable for research applications requiring high specificity and reproducibility.

What are the common applications for HIST1H4A recombinant monoclonal antibodies?

HIST1H4A recombinant monoclonal antibodies are versatile tools in epigenetic research with multiple validated applications. Based on supplier specifications and research practices, common applications include:

Most HIST1H4A antibodies show cross-reactivity across multiple species, including human, mouse, and rat, due to the high conservation of histone H4 across species . This makes them valuable tools for comparative studies across different model organisms.

How can HIST1H4A recombinant monoclonal antibodies be used to study histone post-translational modifications?

Histone H4 undergoes various post-translational modifications (PTMs) that regulate chromatin structure and gene expression. HIST1H4A recombinant monoclonal antibodies can be used to investigate these modifications through several sophisticated approaches:

• Modification-specific antibodies: While pan-histone H4 antibodies recognize the histone regardless of modification state, researchers can select modification-specific antibodies that recognize particular PTMs such as acetylation at lysine 5 (H4K5ac), methylation at lysine 20 (H4K20me), or other specific modifications . This allows for the precise mapping of modification patterns across the genome.

• Sequential ChIP (Re-ChIP): This advanced technique involves:

  • First immunoprecipitation with a pan-histone H4 antibody

  • Elution of the precipitated chromatin

  • Second immunoprecipitation with a modification-specific antibody

  • Analysis of the resulting DNA to identify regions where both the histone H4 and the specific modification are present

• Mass spectrometry validation: To confirm antibody specificity for histone modifications:

  • Immunoprecipitate histone H4 using the recombinant monoclonal antibody

  • Subject the precipitated material to mass spectrometry analysis

  • Identify the specific modifications present on the immunoprecipitated histones

• ChIP-seq with differential modification analysis: By comparing ChIP-seq data from pan-histone H4 antibodies with modification-specific antibodies, researchers can calculate the proportion of histone H4 molecules bearing specific modifications at each genomic location.

The choice of antibody specificity is critical for these applications, as some antibodies may have cross-reactivity with similar modifications or be affected by neighboring modifications (epitope occlusion) .

What role does HIST1H4A play in enhancing recombinant protein expression, and how can this be studied?

Recent research has revealed that epigenetic regulation, including histone modifications, can significantly impact recombinant protein expression in cell culture systems. The study of HIST1H4A's role in this process involves several approaches:

• Epigenetic modulator studies: Small molecule epigenetic modulators, such as the dual HDAC/LSD1 inhibitor I-4, have been shown to enhance recombinant monoclonal antibody production in Chinese hamster ovary (CHO) cells by affecting histone acetylation and methylation levels . Researchers can investigate:

  • The effect of epigenetic modulators on histone H4 modifications

  • Correlation between H4 modification patterns and recombinant protein expression levels

  • Specific pathways linking H4 modifications to transcriptional activation of recombinant genes

• ChIP-qPCR analysis: This technique can be used to quantify the association of modified histones with recombinant gene sequences:

  • Use HIST1H4A antibodies (both pan and modification-specific) to immunoprecipitate chromatin

  • Perform qPCR with primers specific to the recombinant gene sequence

  • Quantify the enrichment of specific histone marks at the recombinant gene locus

• Gene interference studies: Researchers have found that interference with the HDAC5 gene increased monoclonal antibody titer by 1.64-fold . Similar approaches can be used to study how modulation of histone H4 and its modifications affects recombinant protein expression:

  • siRNA or CRISPR-based targeting of histone modifying enzymes

  • Correlation of changes in H4 modification patterns with recombinant protein expression

  • Investigation of specific transcription factors recruited by modified H4

This research area provides promising strategies for improving biopharmaceutical production through epigenetic engineering.

How can HIST1H4A recombinant monoclonal antibodies be optimized for ChIP-seq applications?

Chromatin immunoprecipitation followed by sequencing (ChIP-seq) is a powerful technique for genome-wide mapping of histone H4 distribution and modifications. Optimizing HIST1H4A antibodies for this application requires careful consideration of several factors:

• Antibody selection and validation:

  • Recombinant monoclonal antibodies offer superior specificity and lot-to-lot consistency compared to polyclonal alternatives

  • Validation should include peptide competition assays to confirm specificity

  • Dot blot assays against modified and unmodified histone peptides help assess cross-reactivity

  • Western blotting of acid-extracted histones confirms recognition of endogenous protein

• Chromatin preparation optimization:

  • Crosslinking conditions (formaldehyde concentration and time) must be optimized for histone H4

  • Sonication parameters should be adjusted to generate fragments of 200-500 bp

  • Pre-clearing of chromatin with protein A/G beads can reduce non-specific binding

• Immunoprecipitation protocol refinement:

  Parameter	Optimization Strategy	Typical Range
  Antibody amount	Titration experiment	2-10 μg per reaction
  Chromatin amount	Optimize signal-to-noise	10-50 μg per reaction
  Incubation time	Balance binding and background	2-16 hours
  Washing stringency	Adjust salt concentration	150-500 mM NaCl
  Elution conditions	Temperature and buffer composition	65°C in 1% SDS

• Sequential ChIP considerations:

  • When studying specific modifications, sequential ChIP with pan-H4 antibody followed by modification-specific antibody improves specificity

  • Elution conditions between ChIP steps must be optimized to prevent antibody carryover

• Library preparation and sequencing:

  • Input normalization is critical for accurate interpretation

  • Spike-in controls (e.g., Drosophila chromatin) can help normalize between experimental conditions

  • Sequencing depth should be sufficient to capture the distribution pattern (typically 20-50 million reads)

These optimizations help ensure high-quality ChIP-seq data when using HIST1H4A recombinant monoclonal antibodies.

What are the key factors affecting specificity when using HIST1H4A antibodies?

The specificity of HIST1H4A antibodies is influenced by multiple factors that researchers must carefully control:

• Antibody format and production method:

  • Recombinant monoclonal antibodies typically offer superior specificity compared to polyclonal antibodies

  • The expression system used for antibody production can affect glycosylation patterns and folding, potentially impacting specificity

  • Clone selection during antibody development significantly influences epitope recognition

• Cross-reactivity with other histone variants:

  • Histone H4 is highly conserved, with multiple gene family members (H4/A, H4/B, H4/C, etc.)

  • Most HIST1H4A antibodies will recognize multiple H4 variants due to sequence similarity

  • If variant-specific detection is required, epitopes must be carefully selected from divergent regions

• Impact of post-translational modifications:

  • Modifications near the antibody epitope can block binding (epitope masking)

  • Pan-histone H4 antibodies should ideally recognize H4 regardless of modification state

  • Modification-specific antibodies must be validated against panels of modified peptides to confirm specificity

• Fixation and sample preparation effects:

  Sample Preparation Method	Impact on Epitope Accessibility	Recommended Antibody Dilution Adjustment
  Formaldehyde fixation	May mask some epitopes	Increase concentration by 2-5×
  Methanol fixation	Better for some histone epitopes	Standard concentration
  Heat-induced epitope retrieval	Improves accessibility in FFPE samples	May allow reduced concentration
  Native (non-crosslinked) ChIP	Maintains natural epitope structure	Lower concentration, gentler washing

• Validation strategies to confirm specificity:

  • Peptide competition assays

  • Western blotting in wild-type vs. HIST1H4A-depleted cells

  • Testing with recombinant H4 variants

  • Mass spectrometry analysis of immunoprecipitated material

By carefully considering these factors, researchers can ensure optimal specificity when using HIST1H4A antibodies for various applications.

How can researchers troubleshoot common issues with HIST1H4A antibodies in Western blotting experiments?

Western blotting with HIST1H4A antibodies presents unique challenges due to the small size and highly basic nature of histone proteins. Here are methodological solutions for common issues:

• Poor detection or weak signal:

  • Problem: Histones are small (11.4 kDa for H4) and can be lost during transfer

  • Solution: Use specialized transfer conditions:

    • Transfer buffer with low (10-15%) methanol content

    • PVDF membrane instead of nitrocellulose (better retention of small proteins)

    • Extended transfer time (1-2 hours) at lower voltage (30-50V)

    • Consider semi-dry transfer systems for more efficient transfer of small proteins

• Multiple bands or unexpected molecular weight:

  • Problem: Detection of degradation products or modified forms

  • Solution:

    • Use acid extraction methods specific for histones

    • Include protease and phosphatase inhibitors during extraction

    • Consider using specialized histone extraction kits

    • Run appropriate molecular weight markers for low MW proteins

• High background:

  • Problem: Non-specific binding due to basic nature of histones

  • Solution:

    • Increase blocking time and concentration (5% BSA often works better than milk)

    • Use casein-based blockers which work well for basic proteins

    • Add 0.1-0.5% Triton X-100 to washing buffer

    • Optimize antibody concentration (typically 1:1000-1:5000 dilution)

• Inconsistent loading control issues:

  • Problem: Traditional loading controls like GAPDH have different extraction efficiency than histones

  • Solution:

    • Use total protein staining methods (Ponceau S, SYPRO Ruby) before immunoblotting

    • Use other core histones as loading controls

    • Consider specialized loading controls for nuclear proteins

• Specialized techniques for histone Western blotting:

  • Consider AU-PAGE (acid-urea polyacrylamide gel electrophoresis) for better separation of histone variants and modified forms

  • Use Triton-Acid-Urea (TAU) gels to separate histones based on both size and charge

  • For quantitative analysis, consider fluorescent secondary antibodies rather than chemiluminescence

These methodological approaches can significantly improve the quality and reproducibility of Western blotting experiments with HIST1H4A antibodies.

What are the optimal conditions for using HIST1H4A antibodies in immunofluorescence and immunohistochemistry applications?

Successful immunofluorescence (IF) and immunohistochemistry (IHC) experiments with HIST1H4A antibodies require careful optimization of multiple parameters:

• Fixation optimization:

  • Paraformaldehyde (PFA) fixation:

    • Standard: 4% PFA for 10-15 minutes at room temperature

    • For better nuclear penetration: Add 0.1-0.5% Triton X-100 to fixative

    • Post-fixation permeabilization: 0.1-0.2% Triton X-100 for 5-10 minutes

  • Methanol fixation:

    • Often superior for nuclear antigens like histones

    • 100% ice-cold methanol for 10 minutes at -20°C

    • No additional permeabilization needed

• Antigen retrieval methods for IHC:

  Method	Protocol	Best For
  Heat-induced epitope retrieval (HIER)	10mM sodium citrate buffer pH 6.0, 95-100°C for 20 minutes	FFPE tissue sections
  Enzymatic retrieval	Proteinase K (10-20 μg/ml) for 10-15 minutes at 37°C	Some frozen sections
  Pressure cooker method	Sodium citrate buffer, full pressure for 3 minutes	Difficult epitopes in FFPE tissues

• Blocking optimization:

  • 5-10% normal serum (matching secondary antibody species) in PBS

  • Addition of 0.1-0.3% Triton X-100 for nuclear penetration

  • 1-2% BSA to reduce non-specific binding

  • 0.1-0.2% gelatin can help reduce background with some antibodies

• Antibody incubation parameters:

  • Primary antibody dilution: Typically 1:100-1:500 for IF/IHC applications

  • Incubation time and temperature options:

    • 1-2 hours at room temperature, or

    • Overnight at 4°C (often gives better signal-to-noise ratio)

  • Diluent composition: PBS with 1% BSA and 0.1% Triton X-100

• Special considerations for dual/multiple labeling:

  • When combining with other histone modification antibodies:

    • Use antibodies raised in different host species

    • If same host species is necessary, use directly conjugated antibodies

    • Consider sequential staining protocols with complete blocking between rounds

• Counterstaining and mounting:

  • DAPI or Hoechst 33342 for nuclear counterstaining (1 μg/ml for 5-10 minutes)

  • Use anti-fade mounting media containing DAPI for long-term storage

  • Consider hardset mounting media for confocal microscopy

• Controls for validation:

  • Positive control (tissues/cells known to express histone H4)

  • Negative control (primary antibody omission)

  • Peptide competition control to confirm specificity

  • siRNA knockdown controls (not applicable for histones, but genetic models with tagged histones can be used)

These optimized conditions will help ensure specific and reproducible detection of histone H4 in IF and IHC applications while minimizing background and non-specific staining.

How can researchers validate the specificity and efficiency of HIST1H4A antibodies for ChIP experiments?

Validation of HIST1H4A antibodies for ChIP applications is critical for generating reliable and reproducible results. A comprehensive validation approach includes:

• Pre-experimental validation:

  • Western blotting: Confirm that the antibody recognizes a single band of the correct size (11.4 kDa)

  • Dot blot analysis: Test against modified and unmodified peptides to assess specificity

  • Immunofluorescence: Verify nuclear localization consistent with histone distribution

• ChIP-specific validation experiments:

  • Peptide competition ChIP: Perform parallel ChIP reactions with and without competing peptide

  • Serial dilution ChIP: Establish the linear range of antibody-chromatin interaction

  • Analysis of known target regions: Test enrichment at constitutively expressed genes

  • Negative control regions: Confirm lack of enrichment at silent genes or gene deserts

• Quantitative assessment of ChIP efficiency:

  Validation Parameter	Experimental Approach	Acceptance Criteria
  Enrichment efficiency	qPCR of positive control regions	>5-fold over background
  Signal-to-noise ratio	Compare to IgG control	>10-fold signal over IgG
  Specificity	Peptide competition	>80% signal reduction
  Reproducibility	Technical replicates	CV <20%
  Sensitivity	Titration of antibody amount	Consistent enrichment pattern

• Genome-wide validation for ChIP-seq:

  • Fragment size distribution: Ensure proper chromatin fragmentation (200-500 bp)

  • Library complexity assessment: >10 million unique reads

  • Peak distribution analysis: Histone H4 should show broad distribution

  • Correlation with known marks: H4 distribution should correlate with open chromatin

• Cross-validation approaches:

  • Antibody comparison: Test multiple antibody clones against the same target

  • Orthogonal techniques: Compare results with CUT&RUN or CUT&Tag methods

  • Tagged histone systems: When possible, compare with results from epitope-tagged H4

• Advanced validation for modification-specific antibodies:

  • Mass spectrometry validation of immunoprecipitated material

  • Sequential ChIP with pan-H4 antibody followed by modification-specific antibody

  • Use of cells with mutations in the corresponding histone modifying enzymes

Through these validation approaches, researchers can ensure that HIST1H4A antibodies used in ChIP experiments are specific, sensitive, and provide reproducible results.