HIST1H4A (Ab-35) Antibody

What is HIST1H4A and what biological functions does it serve?

HIST1H4A is one of multiple genes encoding histone H4, a core component of the nucleosome which serves as the basic building block of chromatin. Histone H4 is essential for packaging DNA into chromatin, thereby regulating DNA accessibility to the cellular machinery involved in transcription, replication, and repair processes. This protein plays a central role in chromatin structure and dynamics, with its functions primarily regulated through post-translational modifications .

Histone H4 is highly conserved and participates in multiple critical cellular processes including:

• DNA compaction and chromatin organization

• Regulation of gene expression through accessibility control

• DNA replication and repair mechanisms

• Maintenance of chromosomal stability

• Epigenetic inheritance patterns

The protein has numerous aliases including H4/A, H4/B, H4/C, and several others, reflecting its importance across different genetic loci and conserved structure .

What applications is the HIST1H4A (Ab-35) antibody validated for?

The HIST1H4A (Ab-35) polyclonal antibody has been validated for multiple research applications:

The antibody specifically recognizes the region around arginine 35 of human histone H4, making it particularly useful for studies investigating specific modifications at this site .

How does HIST1H4A differ from other histone H4 variants?

HIST1H4A is one of multiple genes encoding histone H4, which is among the most conserved proteins in eukaryotes. While the protein sequence of histone H4 is highly conserved across its various encoding genes (HIST1H4A through HIST1H4L, HIST2H4A, HIST2H4B, etc.), these variants differ primarily in their:

• Genomic locations and organization within histone clusters

• Regulatory elements controlling their expression

• Expression patterns during the cell cycle

• Responsiveness to cellular signals

What are the optimal protocols for using HIST1H4A (Ab-35) antibody in chromatin immunoprecipitation (ChIP) experiments?

For optimal ChIP experiments using HIST1H4A (Ab-35) antibody, the following methodology is recommended:

• Cross-linking and Cell Preparation:

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

  • Quench with 125 mM glycine for 5 minutes

  • Wash cells twice with cold PBS

  • Collect 1-5×10^6 cells by centrifugation

• Chromatin Preparation:

  • Lyse cells in appropriate buffer containing protease inhibitors

  • Sonicate chromatin to generate 200-500 bp fragments

  • Centrifuge to remove debris and transfer supernatant to new tube

• Immunoprecipitation:

  • Pre-clear chromatin with protein A/G beads

  • Add HIST1H4A (Ab-35) antibody (2-5 μg per reaction)

  • Incubate overnight at 4°C with rotation

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

  • Wash beads with increasing stringency buffers

• DNA Recovery and Analysis:

  • Reverse cross-links at 65°C overnight

  • Treat with RNase A and Proteinase K

  • Purify DNA using column purification

  • Analyze by qPCR, sequencing, or other appropriate methods

This protocol has been validated in studies examining histone modifications related to gene expression regulation, such as those investigating HAT1's role in coordinating histone production and acetylation .

How should researchers design control experiments when using the HIST1H4A (Ab-35) antibody?

Proper control experiments are essential for validating results obtained with HIST1H4A (Ab-35) antibody:

• Positive Controls:

  • Use HeLa acid extract as a positive control sample, which contains abundant histone H4

  • Include analysis of known regions where histone H4 is expected to be present (such as actively transcribed genes for acetylated forms)

  • For western blots, compare samples treated with histone deacetylase inhibitors (like sodium butyrate) against untreated samples

• Negative Controls:

  • Include isotype control antibody (rabbit IgG) matching the host species and isotype of the primary antibody

  • Analyze genomic regions known to lack histone H4 modifications of interest

  • Include samples where the target protein is depleted (RNAi knockdown when possible)

• Antibody Validation Controls:

  • Peptide competition assay using the immunogen peptide (amino acids around Arg35 of human histone H4)

  • Demonstrate specificity through western blot showing the appropriate 11-15 kDa band

  • Compare results with other validated histone H4 antibodies targeting different epitopes

These control experiments help distinguish specific signals from background and confirm antibody specificity, which is particularly important in epigenetic studies where cross-reactivity with similar histone variants can complicate interpretation .

What is the recommended procedure for using HIST1H4A (Ab-35) antibody in immunofluorescence studies?

For optimal immunofluorescence results with HIST1H4A (Ab-35) antibody, follow this procedure:

• Cell Preparation:

  • Culture cells on coverslips to 70-80% confluence

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

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

• Blocking and Antibody Incubation:

  • Block with 1-5% BSA in PBS for 1 hour at room temperature

  • Dilute HIST1H4A (Ab-35) antibody in blocking solution (start with 1:100 dilution and optimize)

  • Incubate with primary antibody overnight at 4°C

  • Wash 3 times with PBS, 5 minutes each

  • Incubate with fluorophore-conjugated secondary antibody (anti-rabbit) for 1 hour at room temperature

  • Wash 3 times with PBS, 5 minutes each

• Nuclear Counterstaining and Mounting:

  • Counterstain nuclei with DAPI (0.1-1 μg/mL) for 5 minutes

  • Wash briefly with PBS

  • Mount coverslips using appropriate mounting medium

• Imaging and Analysis:

  • Capture images using confocal or fluorescence microscopy

  • For co-localization studies, use appropriate filter sets for multiple fluorophores

  • Analyze nuclear staining patterns, noting distribution patterns

As demonstrated in reference studies, HIST1H4A should show specific nuclear localization, with distribution patterns that may vary depending on cell cycle stage and treatment conditions . For specialized applications like examining histone modifications in cancer cells, researchers may need to optimize fixation and permeabilization conditions to preserve epitope accessibility.

How can the HIST1H4A (Ab-35) antibody be used to investigate the relationship between histone modifications and gene expression?

The HIST1H4A (Ab-35) antibody provides a powerful tool for investigating histone modifications at arginine 35 and their impact on gene expression through several advanced approaches:

• ChIP-Seq Analysis:

  • Perform ChIP with HIST1H4A (Ab-35) antibody followed by next-generation sequencing

  • Map genome-wide distribution of histone H4 with modified Arg35

  • Correlate modification patterns with gene expression data (RNA-seq)

  • Identify enriched motifs in regions with specific modification patterns

• Sequential ChIP (Re-ChIP):

  • First immunoprecipitate with HIST1H4A (Ab-35) antibody

  • Re-immunoprecipitate with antibodies against other histone marks or transcription factors

  • Identify genomic regions with co-occurrence of multiple modifications

  • Determine how Arg35 modifications interact with other epigenetic marks

• Integration with Transcriptional Activity Assays:

  • Combine ChIP data with nascent transcription assays (e.g., GRO-seq)

  • Use reporter constructs containing promoters of interest

  • Mutate Arg35 to non-modifiable residues and assess transcriptional outcomes

  • Study effects of histone deacetylase inhibitors or other epigenetic modulators

Research has shown that histone H4 modifications are particularly important for coordinating transcriptional responses. For example, HAT1 has been found to coordinate histone production and acetylation, binding specifically to H4 gene promoters through an acetate-sensitive promoter element called the H4-box . This represents a feed-forward regulatory circuit where HAT1 captures acetyl groups on nascent histones and drives H4 production through chromatin binding, supporting proliferation and S-phase progression.

What role does arginine 35 methylation in HIST1H4A play in epigenetic regulation?

Arginine 35 methylation on histone H4 represents an important but less studied modification compared to lysine methylations. Based on current research:

• Functional Significance:

  • Arginine methylation at position 35 can exist in different states: monomethylation, symmetric dimethylation, or asymmetric dimethylation

  • These modifications influence chromatin structure and recruitment of specific reader proteins

  • They may antagonize or synergize with nearby modifications, creating a complex "histone code"

  • Arg35 methylation appears to play roles in both gene activation and repression, depending on cellular context

• Regulatory Mechanisms:

  • Specific protein arginine methyltransferases (PRMTs) target Arg35

  • Arginine demethylases can remove these marks in response to specific signals

  • The balance between methylation and demethylation creates dynamic regulation

  • Crosstalk with nearby modifications, particularly acetylation at lysines, affects recognition and function

• Disease Relevance:

  • Aberrant arginine methylation patterns have been observed in various cancers

  • Viral infections can modulate histone arginine methylation to promote viral reactivation

  • For example, KSHV-encoded ORF59 has been shown to modulate histone arginine methylation of the viral genome

The HIST1H4A (Ab-35) antibody specifically recognizing the region around Arg35 enables researchers to investigate these mechanisms in detail, particularly when used in conjunction with modification-specific antibodies.

How can HIST1H4A (Ab-35) antibody be used in studies of cell cycle regulation and chromatin dynamics?

The HIST1H4A (Ab-35) antibody can be leveraged for sophisticated studies of cell cycle-dependent chromatin dynamics through several approaches:

• Cell Cycle Synchronization Studies:

  • Synchronize cells at different cell cycle phases using methods like double thymidine block or nocodazole treatment

  • Perform ChIP or immunofluorescence at defined time points using HIST1H4A (Ab-35) antibody

  • Track changes in histone H4 modifications throughout the cell cycle

  • Correlate with DNA replication timing and transcriptional activity

• Live Cell Imaging with Proximity Ligation Assays:

  • Combine HIST1H4A (Ab-35) antibody with antibodies against cell cycle regulators

  • Perform proximity ligation assays to detect interactions in situ

  • Use microscopy techniques as demonstrated in reference studies to capture changes in protein interactions over time

  • Correlate with cell cycle markers and progression indicators

• Analysis of Chromatin Assembly Pathways:

  • Study incorporation of newly synthesized histones during S-phase

  • Investigate the timing of specific post-translational modifications

  • Examine the role of histone chaperones and assembly factors

  • Track arginine 35 modifications in relation to DNA replication

Research has demonstrated that HAT1 expression is critical for S-phase progression and maintenance of histone H3 lysine 9 acetylation at proliferation-associated genes, including histone genes themselves . Cells depleted of HAT1 show accumulation in G1 phase (approximately 5.2% increase compared to control), suggesting an important role in cell cycle progression. The HIST1H4A (Ab-35) antibody can help elucidate how arginine 35 modifications contribute to these regulatory networks.

What are common pitfalls when using HIST1H4A (Ab-35) antibody and how can they be addressed?

When working with HIST1H4A (Ab-35) antibody, researchers may encounter several common challenges:

• Cross-Reactivity Issues:

  • Problem: The antibody may cross-react with other histone H4 variants due to high sequence conservation.

  • Solution: Use peptide competition assays with the specific immunogen (peptide sequence around Arg35) to confirm specificity. Compare results with knockout or knockdown controls when available.

• Epitope Masking:

  • Problem: Post-translational modifications near Arg35 may mask the epitope and prevent antibody binding.

  • Solution: Use multiple antibodies targeting different regions of histone H4. Consider native ChIP methods for certain applications to preserve natural modification states.

• Fixation-Related Issues:

  • Problem: Overfixation can reduce epitope accessibility in IHC and IF applications.

  • Solution: Optimize fixation conditions (time, temperature, fixative concentration). Consider epitope retrieval methods for tissue samples, such as heat-induced or enzymatic retrieval.

• Signal-to-Noise Ratio Problems:

  • Problem: High background or weak specific signal in immunoassays.

  • Solution: Optimize blocking conditions (use 5% BSA or normal serum from the secondary antibody host species). Titrate antibody concentrations carefully, starting with the manufacturer's recommended dilutions (1:10-1:100 for IHC) .

• Storage and Handling Issues:

  • Problem: Antibody degradation affecting performance.

  • Solution: Store properly (often at -20°C or -80°C with 50% glycerol, 0.01M PBS, pH 7.4, and 0.03% Proclin 300 as preservative) . Avoid repeated freeze-thaw cycles by making small aliquots upon receipt.

How should researchers interpret conflicting data from HIST1H4A (Ab-35) antibody experiments?

When faced with conflicting results in HIST1H4A (Ab-35) antibody experiments, a systematic approach to data interpretation is essential:

• Antibody Validation Assessment:

  • Confirm antibody lot consistency and validate each new lot

  • Verify specificity through western blot against purified histones or nuclear extracts

  • Use peptide competition assays to confirm epitope specificity

  • Compare with results from alternative antibodies targeting the same or nearby epitopes

• Experimental Variables Analysis:

  • Document all variables between conflicting experiments (cell types, growth conditions, fixation methods)

  • Consider cell cycle differences, as histone modifications vary throughout cell cycle phases

  • Evaluate effects of culture conditions on histone modification states

  • Assess potential technical variations in ChIP efficiency or antibody incubation conditions

• Reconciliation Strategies:

  • Use orthogonal methods to verify findings (e.g., compare ChIP results with mass spectrometry data)

  • Perform dose-response or time-course experiments to identify optimal conditions

  • Consider single-cell techniques to address potential heterogeneity in cell populations

  • Use genetic approaches (CRISPR/Cas9) to validate antibody specificity through targeted mutation of arginine 35

• Data Integration Approaches:

  • Look for consistent patterns across different experimental systems

  • Weigh results based on strength of controls and methodological rigor

  • Consider biological context and relevance to the research question

  • Use computational approaches to integrate multiple datasets and identify robust signals

What are the latest advanced techniques for studying histone modifications using HIST1H4A (Ab-35) antibody?

Cutting-edge techniques for studying histone modifications using HIST1H4A (Ab-35) antibody include:

• CUT&RUN and CUT&Tag:

  • More sensitive alternatives to traditional ChIP

  • Use targeted nuclease activity to cleave DNA specifically at antibody-bound sites

  • Require fewer cells and less antibody than conventional ChIP

  • Provide improved signal-to-noise ratio for studying arginine 35 modifications

• Single-Cell ChIP-Seq:

  • Analyze histone modifications at the single-cell level

  • Reveal cell-to-cell variation in histone modification patterns

  • Identify rare cell populations with distinct epigenetic signatures

  • Map changes during cellular differentiation or disease progression

• Mass Spectrometry Integration:

  • Combine antibody-based enrichment with mass spectrometry analysis

  • Identify co-occurring modifications on the same histone molecule

  • Quantify relative abundance of different modification states

  • Discover novel modifications or combinations affecting Arg35 function

• CRISPR-Based Epigenome Editing:

  • Use dCas9 fused to histone modifiers to manipulate Arg35 modifications at specific genomic loci

  • Compare results with antibody-based detection of natural modification patterns

  • Establish causal relationships between specific modifications and gene expression

  • Create synthetic epigenetic states to test mechanistic hypotheses

• Microfluidic Approaches:

  • Combine antibody-based detection with microfluidic devices

  • Perform high-throughput analysis of histone modifications

  • Study dynamic changes in modification patterns over time

  • Integrate with other genomic and proteomic analyses

These advanced techniques allow researchers to move beyond correlation to establish causal relationships between histone modifications at Arg35 and functional outcomes, providing deeper insights into the biological roles of HIST1H4A modifications in health and disease .

How does HIST1H4A modification pattern change in cancer cells and how can the antibody be used in cancer research?

HIST1H4A modifications, particularly at Arg35, undergo significant changes in cancer cells that can be studied using the HIST1H4A (Ab-35) antibody:

• Cancer-Specific Modification Patterns:

  • Altered arginine methylation patterns correlate with specific cancer types

  • Changes in the balance between different histone H4 modifications affect gene expression programs

  • Cancer cells often show global hypoacetylation with localized hyperacetylation at oncogenes

  • The HIST1H4A (Ab-35) antibody can map these changes across the genome

• Research Applications in Cancer Models:

  • Compare histone H4 modification patterns between normal and cancerous tissues

  • Correlate modifications with tumor stage, grade, and patient outcomes

  • Study effects of epigenetic drugs (HDAC inhibitors, PRMT inhibitors) on Arg35 modification state

  • Investigate cancer-specific epigenetic vulnerabilities through ChIP-seq profiling

• Experimental Approaches:

  • Use the antibody in cancer cell lines treated with epigenetic modulators

  • Apply to patient-derived xenograft models to track modification changes during treatment

  • Perform IHC on tissue microarrays for biomarker discovery

  • Integrate with genomic mutation data to identify relationships between genetic and epigenetic alterations

Research has demonstrated that histone H4 acetylation can be detected in cancer cell lines like HeLa cells, with modifications responding to treatments such as sodium butyrate (a histone deacetylase inhibitor) . These techniques are valuable for understanding how epigenetic dysregulation contributes to cancer development and progression.

What insights can be gained by studying HIST1H4A modifications in neurodegenerative diseases?

The study of HIST1H4A modifications in neurodegenerative diseases offers important insights into disease mechanisms and potential therapeutic targets:

• Neurodegenerative Disease Relevance:

  • Histone modifications regulate neuronal gene expression programs crucial for brain function

  • Arginine methylation affects expression of genes involved in neuronal survival and function

  • Age-related changes in histone modification patterns may contribute to neurodegenerative processes

  • Aberrant HIST1H4A modifications have been associated with memory formation and cognitive decline

• Experimental Approaches:

  • Use HIST1H4A (Ab-35) antibody in ChIP-seq studies of brain tissue from disease models

  • Compare histone modification patterns in affected vs. unaffected brain regions

  • Study temporal changes in modifications during disease progression

  • Investigate effects of neuroprotective compounds on histone H4 modification state

• Therapeutic Implications:

  • Identify potential epigenetic targets for intervention

  • Develop biomarkers for disease progression based on modification patterns

  • Evaluate efficacy of epigenetic modulators in reversing disease-associated modifications

  • Design targeted approaches to restore normal histone modification patterns

• Integration with Other Data Types:

  • Correlate histone modifications with transcriptome changes in diseased tissue

  • Connect genetic risk factors with epigenetic dysregulation

  • Combine with proteomics data to understand broader chromatin remodeling events

  • Relate to cellular phenotypes in patient-derived neurons or organoids

While the search results don't specifically address neurodegenerative diseases, the fundamental role of histone H4 in transcriptional regulation makes its modification patterns highly relevant to understanding neuronal gene expression changes in these conditions.

How can HIST1H4A (Ab-35) antibody be integrated with spatial transcriptomics for epigenomic analysis?

Integrating HIST1H4A (Ab-35) antibody with spatial transcriptomics represents an emerging frontier in epigenomic research:

• Technical Integration Approaches:

  • Combine immunofluorescence using HIST1H4A (Ab-35) antibody with spatial transcriptomics platforms

  • Perform sequential immunostaining and RNA detection on the same tissue sections

  • Develop computational methods to integrate histone modification data with spatially resolved gene expression

  • Use multi-modal approaches to simultaneously detect histone modifications and RNA in situ

• Experimental Design Considerations:

  • Optimize tissue preparation protocols to preserve both epitope and RNA integrity

  • Select anatomical regions of interest with known biological heterogeneity

  • Include relevant cellular markers to identify specific cell types

  • Design appropriate controls to account for technical variation across modalities

• Analytical Frameworks:

  • Develop statistical methods to correlate spatial patterns of histone modifications with gene expression

  • Create visualization tools to represent multi-modal spatial data

  • Apply machine learning approaches to identify spatial domains with distinct epigenetic signatures

  • Integrate with single-cell approaches for comprehensive epigenetic profiling

• Biological Applications:

  • Map tissue-specific epigenetic regulatory domains in development and disease

  • Identify local microenvironmental effects on histone modification patterns

  • Study epigenetic heterogeneity within complex tissues like tumors or brain

  • Investigate spatial relationships between cells with different epigenetic states

While specific examples using HIST1H4A (Ab-35) antibody in spatial genomics are not directly mentioned in the search results, the antibody's validated use in immunohistochemistry and immunofluorescence makes it a suitable candidate for such advanced spatial applications.

What are the future prospects for targeted proteomics approaches using HIST1H4A (Ab-35) antibody?

Targeted proteomics approaches using HIST1H4A (Ab-35) antibody offer promising future directions for histone modification research:

• Antibody-Enhanced Mass Spectrometry:

  • Use HIST1H4A (Ab-35) antibody for immunoprecipitation before MS analysis

  • Enrich for histone H4 protein containing the Arg35 region

  • Identify co-occurring modifications and protein interaction partners

  • Quantify relative abundance of different modification states at and around Arg35

• Proximity Labeling Applications:

  • Develop antibody conjugates with proximity labeling enzymes (BioID, APEX)

  • Map the protein interaction network around modified histones

  • Identify readers, writers, and erasers specific to Arg35-modified histone H4

  • Study dynamic changes in interaction partners under different cellular conditions

• Single-Cell Proteomics Integration:

  • Adapt antibody-based detection for microfluidic single-cell proteomics

  • Profile histone modification heterogeneity at single-cell resolution

  • Correlate with other epigenetic marks and cellular phenotypes

  • Track proteome-wide responses to changes in histone modification state

• Therapeutic Applications:

  • Develop targeted protein degradation approaches (PROTACs) directed at specifically modified histones

  • Use antibody-drug conjugates to target cells with aberrant modification patterns

  • Create screening platforms to identify compounds that modulate specific histone modifications

  • Monitor treatment responses through changes in histone modification profiles