HIST1H3A (Ab-23) Antibody

What is HIST1H3A and why is the acetylation at lysine 23 significant in epigenetic research?

HIST1H3A (Histone Cluster 1, H3a) is a core component of nucleosomes that wrap and compact DNA into chromatin. Nucleosomes play a central role in limiting DNA accessibility to cellular machineries requiring DNA as a template. Histones are critical for transcription regulation, DNA repair, DNA replication, and chromosomal stability .

The acetylation at lysine 23 (acLys23) is a specific post-translational modification that contributes to the histone code, which regulates DNA accessibility. This modification is particularly important because:

• It occurs in the globular domain of histone H3

• It influences nucleosome stability

• It serves as a marker for transcriptionally active chromatin regions

• It interacts with specific reader proteins that mediate downstream effects

This specific modification allows researchers to track particular chromatin states associated with gene expression and epigenetic regulation.

What applications has the HIST1H3A (Ab-23) antibody been validated for?

The HIST1H3A (Ab-23) antibody has been rigorously validated across multiple applications:

The antibody is particularly valuable for chromatin immunoprecipitation studies, making it an essential tool for researchers investigating histone modifications and their role in gene regulation .

How does HIST1H3A (Ab-23) antibody differ from other histone H3 modification-specific antibodies?

The HIST1H3A (Ab-23) antibody specifically recognizes the acetylation at lysine 23 on histone H3.1, distinguishing it from antibodies targeting other modifications such as:

• H3K27me3 (trimethylation at lysine 27), which is associated with gene silencing

• H3K4me3 (trimethylation at lysine 4), which is associated with active promoters

• H3K9me3 (trimethylation at lysine 9), which is associated with heterochromatin

• H3K36me3 (trimethylation at lysine 36), which is enriched in actively transcribed regions

This specificity is crucial as different modifications can have opposing effects on chromatin structure and gene expression. The antibody was developed using a peptide sequence specifically around the site of Lys (23) derived from Human Histone H3.1, ensuring high selectivity for this particular modification .

What are the optimal conditions for using HIST1H3A (Ab-23) antibody in ChIP experiments?

For optimal ChIP results with HIST1H3A (Ab-23) antibody, follow these guidelines:

Chromatin Preparation:

• Cross-link cells with 1% formaldehyde for 10 minutes at room temperature

• Quench with 125 mM glycine for 5 minutes

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

• Verify fragment size by agarose gel electrophoresis

Immunoprecipitation:

• Use 2-5 μg of HIST1H3A (Ab-23) antibody per ChIP reaction

• Incubate with chromatin overnight at 4°C with rotation

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

• Perform stringent washing steps to reduce background

• Elute bound chromatin and reverse cross-links

Controls:

• Include a non-specific IgG control

• Use input chromatin (pre-immunoprecipitation) as a reference

• Consider including a positive control targeting a highly abundant histone mark

This protocol has been validated in human cell lines including HeLa and U2OS, showing specific enrichment at transcriptionally active regions .

How should sample preparation be optimized for Western blotting with HIST1H3A (Ab-23) antibody?

For successful Western blotting with HIST1H3A (Ab-23) antibody:

Histone Extraction:

• Use acid extraction methods (e.g., 0.2N HCl or triton extraction) to isolate histones

• Include HDAC inhibitors (e.g., sodium butyrate, TSA) in lysis buffers to preserve acetylation marks

• Add protease inhibitors to prevent degradation

• Include phosphatase inhibitors if examining phosphorylation marks simultaneously

Gel Electrophoresis and Transfer:

• Use 15-18% SDS-PAGE gels for optimal histone separation

• Consider using Triton-Acid-Urea (TAU) gels for better resolution of modified histones

• Transfer to PVDF membranes at low voltage (30V) overnight for efficient transfer of small proteins

Antibody Incubation:

• Block with 5% BSA (not milk, which contains bioactive proteins)

• Use antibody at 1:500 to 1:2000 dilution

• Incubate overnight at 4°C for best results

Detection:

• Use ECL or fluorescent secondary antibodies for detection

• Expected band size is approximately 15-17 kDa

This approach has been validated on human and mouse cell lines and tissue samples, showing specific detection of H3K23ac modification .

What considerations are important when using HIST1H3A (Ab-23) antibody for immunofluorescence?

For optimal immunofluorescence with HIST1H3A (Ab-23) antibody:

Fixation Methods:

• 4% paraformaldehyde (10 minutes at room temperature) preserves most epitopes

• Methanol fixation (-20°C for 10 minutes) may better expose some nuclear epitopes

• Avoid over-fixation, which can mask the epitope

Permeabilization:

• Use 0.1-0.5% Triton X-100 for nuclear antigens

• Include a permeabilization step (10 minutes at room temperature)

Antigen Retrieval:

• Heat-mediated antigen retrieval with citrate buffer (pH 6.0) may improve signal

• For tissue sections, enzymatic retrieval using proteinase K can be effective

Antibody Incubation:

• Dilute antibody 1:100 to 1:500 in blocking buffer

• Incubate overnight at 4°C for best results

• Consider adding 0.1% Tween-20 to reduce background staining

Controls:

• Include a negative control (primary antibody omitted)

• Consider a peptide competition assay to validate specificity

Researchers have successfully used these conditions for detecting H3K23ac in human cell lines including HeLa and 22Rv1, with strong nuclear staining patterns corresponding to euchromatic regions .

How can I distinguish between true H3K23ac signals and non-specific binding in my experiments?

To ensure signal specificity when using HIST1H3A (Ab-23) antibody:

Validation Approaches:

• Peptide Competition Assay: Pre-incubate antibody with excess H3K23ac peptide before use. True signals should disappear.

• Knockout/Knockdown Controls: Use cells where the H3 acetyltransferase responsible for K23 acetylation is depleted.

• HDAC Inhibition: Treatment with HDAC inhibitors should increase global H3K23ac levels.

• Correlation With Known Marks: H3K23ac should positively correlate with other active chromatin marks (H3K4me3, H3K27ac).

Common False Positives:

• Cross-reactivity with other acetylated lysines on H3 (K18, K27)

• Non-specific binding to highly charged nuclear proteins

• Inadequate blocking leading to high background

Data Analysis Considerations:

• In ChIP-seq, compare enrichment patterns with published datasets

• For imaging, quantify nuclear/cytoplasmic signal ratios

• For Western blots, verify molecular weight and compare with positive controls

When properly validated, H3K23ac signals should show nuclear localization and enrichment at actively transcribed genes and enhancers .

What are the most common issues encountered with HIST1H3A (Ab-23) antibody and how can they be resolved?

These troubleshooting approaches have been validated across multiple research settings and can significantly improve experimental outcomes when working with this antibody .

How does the dynamic nature of histone acetylation affect experimental design with HIST1H3A (Ab-23) antibody?

Histone acetylation is highly dynamic, which creates several experimental considerations:

Temporal Factors:

• Acetylation patterns can change rapidly (within minutes) in response to stimuli

• Consider time-course experiments to capture dynamics

• Synchronize cells when studying cell cycle-dependent changes

• The half-life of H3K23ac may vary between cell types and conditions

Environmental Considerations:

• Serum starvation can alter global acetylation patterns

• Cell density affects histone modification levels

• Stress responses can rapidly change the epigenetic landscape

• Consider controlling for circadian effects in animal studies

Experimental Approaches:

• Use HDAC inhibitors as positive controls for increased acetylation

• Consider pulse-chase experiments with labeled histones

• For ChIP-seq, validate findings with orthogonal methods (e.g., CUT&RUN)

Research has shown that H3.3 deposition and modification, which includes K23 acetylation, can be induced by stimuli such as interferon and remains stable in non-dividing cells while being diminished in dividing cells . This dynamic nature means experimental conditions must be carefully controlled and timed to ensure reproducibility.

How can HIST1H3A (Ab-23) antibody be used to study the relationship between histone variants and modifications?

The HIST1H3A (Ab-23) antibody can be leveraged to investigate the complex interplay between histone variants and their modifications:

Sequential ChIP Approaches:

• Perform ChIP-reChIP with antibodies against histone variants (H3.1, H3.3) followed by H3K23ac

• This can reveal whether K23 acetylation preferentially occurs on specific H3 variants

Mass Spectrometry Integration:

• Use the antibody for immunoprecipitation followed by mass spectrometry

• This allows identification of co-occurring modifications on the same histone tail

• Can reveal "modification signatures" specific to certain genomic regions

Genomic Approaches:

• Combine ChIP-seq for H3K23ac with other techniques like ATAC-seq

• This provides insights into chromatin accessibility in relation to this modification

• Correlation with transcriptomic data (RNA-seq) can reveal functional impacts

Research has shown that H3.3 variant carries higher levels of active marks like K36me3 compared to H3.1, and similar patterns may exist for K23ac . Understanding these relationships is crucial for deciphering the histone code and its biological implications.

What role does H3K23 acetylation play in cell cycle regulation and DNA damage response?

H3K23 acetylation has emerging roles in cell cycle and DNA damage pathways:

Cell Cycle Dynamics:

• H3.1 deposition occurs primarily during S-phase, but modifications like K23ac show cell-cycle dependent patterns

• p53 ensures normal behavior and modification of H3.1 during G1/S transition

• HIST1H3A (Ab-23) antibody can track these changes through cell cycle phases

• Different from H3.3, which is deposited throughout the cell cycle independent of replication

DNA Damage Response:

• H3K23ac levels change in response to genotoxic stress

• May function as a platform for recruitment of DNA repair factors

• Can be studied using HIST1H3A (Ab-23) antibody before and after damage induction

• Often works in concert with other modifications like H3S10 phosphorylation

Experimental Approaches:

• Synchronize cells at different cell cycle stages and analyze H3K23ac patterns

• Induce DNA damage with agents like neocarzinostatin or UV and track H3K23ac changes

• Combine with cell cycle markers (e.g., PCNA for S-phase) in co-localization studies

These applications allow researchers to understand how this specific modification contributes to maintaining genomic integrity across different cellular states .

How can HIST1H3A (Ab-23) antibody be incorporated into multi-omics approaches for comprehensive epigenetic profiling?

Integrating HIST1H3A (Ab-23) antibody into multi-omics frameworks:

Combined Genomic Approaches:

• ChIP-seq for H3K23ac paired with:

  • ATAC-seq (chromatin accessibility)

  • RNA-seq (transcriptome)

  • DNA methylation analysis (methyl-seq)

  • Chromosome conformation capture (Hi-C)

Single-Cell Applications:

• Single-cell ChIP-seq adaptations for H3K23ac

• CUT&Tag for improved sensitivity in limited samples

• Integration with single-cell RNA-seq for direct correlation with gene expression

Spatial Epigenomics:

• Combine immunofluorescence with RNA-FISH

• Use multiplexed antibody imaging with H3K23ac and other histone marks

• Spatially resolved chromatin profiling technologies

Data Integration Strategies:

• Machine learning approaches to identify epigenetic signatures

• Network analysis to understand regulatory connections

• Trajectory analysis for developmental or disease progression studies

This integrated approach provides a comprehensive understanding of how H3K23 acetylation fits within the broader epigenetic landscape and its functional consequences on gene regulation .

How does the sensitivity and specificity of polyclonal HIST1H3A (Ab-23) antibody compare to monoclonal alternatives?

A detailed comparison between polyclonal HIST1H3A (Ab-23) antibody and monoclonal options:

What emerging technologies will enhance the utility of HIST1H3A (Ab-23) antibody in future epigenetic research?

Emerging technologies poised to expand HIST1H3A (Ab-23) antibody applications:

CUT&Tag and CUT&RUN:

• Higher sensitivity than traditional ChIP

• Requires fewer cells and less antibody

• Better signal-to-noise ratio for H3K23ac detection

• Works with difficult-to-sonicate samples

Proximity Ligation Assays (PLA):

• Detect interactions between H3K23ac and reader proteins

• Visualize spatial relationships between different histone modifications

• Single-molecule resolution of modification patterns

Mass Cytometry (CyTOF):

• Multiplexed analysis of dozens of histone modifications

• Single-cell resolution of epigenetic states

• Metal-conjugated antibodies eliminate spectral overlap issues

Live-Cell Imaging:

• Development of H3K23ac-specific intrabodies

• Real-time tracking of acetylation dynamics

• FRET-based sensors for histone modification changes

Nanopore Sequencing:

• Direct detection of modified nucleosomes

• Long-read epigenetic analysis

• Simultaneous detection of DNA methylation and histone modifications

These technologies will dramatically expand our understanding of H3K23 acetylation dynamics and function in various biological contexts .

How can HIST1H3A (Ab-23) antibody contribute to understanding disease-associated epigenetic dysregulation?

The HIST1H3A (Ab-23) antibody has significant potential in disease research:

Cancer Epigenetics:

• Altered H3K23ac patterns in various cancers

• Association with oncogene activation or tumor suppressor silencing

• Biomarker potential for cancer subtyping and prognosis

• Target for epigenetic therapies (HDAC inhibitors)

Neurodegenerative Disorders:

• Disrupted H3K23ac in Alzheimer's and Parkinson's disease

• Memory formation involves dynamic histone acetylation

• HIST1H3A (Ab-23) antibody can track disease progression in brain tissues

Inflammatory Conditions:

• H3K23ac changes during immune cell activation

• Association with dysregulated inflammation

• Potential for monitoring therapeutic interventions

• Interferon response genes show H3.3 deposition and modification

Research Approaches:

• Patient-derived samples compared to healthy controls

• Animal models of disease progression

• Drug screening for compounds affecting H3K23ac

• Integration with genetic risk factors

Understanding how H3K23 acetylation patterns change in disease states may provide new insights into pathogenesis and identify novel therapeutic targets .