Acetyl-HIST1H4A (K12) Antibody

What is Histone H4 acetylation at lysine 12 and why is it significant?

Histone H4 acetylation at lysine 12 (H4K12ac) represents a specific post-translational modification occurring on the core component of nucleosomes. Nucleosomes wrap and compact DNA into chromatin, limiting DNA accessibility to cellular machineries that require DNA as a template. Histones play a central role in transcription regulation, DNA repair, DNA replication, and chromosomal stability. The accessibility of DNA is regulated through a complex set of post-translational modifications of histones, collectively referred to as the histone code, along with nucleosome remodeling . H4K12 acetylation specifically contributes to creating an open chromatin structure, facilitating transcriptional activation and other DNA-templated processes.

How does H4K12ac function in gene expression regulation?

H4K12 acetylation functions primarily as an activating epigenetic mark that promotes gene expression. When lysine 12 on histone H4 is acetylated, the positive charge of the lysine is neutralized, weakening the interaction between the histone and negatively charged DNA. This modification creates a more open chromatin structure that allows transcription factors and RNA polymerase to access DNA, thereby facilitating gene expression . Additionally, specific proteins with bromodomains can recognize and bind to acetylated histones, further recruiting transcriptional machinery. Research using ChIP-seq with anti-H4K12ac antibodies demonstrates enrichment of this modification at transcriptionally active genomic regions .

What experimental techniques are optimal for studying H4K12ac patterns?

Several techniques are effective for studying H4K12ac patterns in research settings:

• Chromatin Immunoprecipitation (ChIP): ChIP followed by sequencing (ChIP-seq) is the gold standard for genome-wide mapping of H4K12ac. The search results indicate successful ChIP-seq experiments using anti-H4K12ac antibodies with HeLa cells fixed with 1% formaldehyde for 10 minutes, sequenced on the Illumina NovaSeq 6000 to a depth of 60 million reads .

• Immunofluorescence microscopy: For cellular localization studies, paraformaldehyde fixation (4%) with Triton X-100 (0.1%) permeabilization provides optimal results for detecting H4K12ac in nuclear regions .

• Western blotting: Effective for quantitative assessment of H4K12ac levels in different experimental conditions, particularly when comparing treatment effects such as histone deacetylase inhibition with Trichostatin A .

• Flow cytometry: Enables analysis of H4K12ac levels in individual cells, particularly useful for heterogeneous populations or examining effects of treatments at the single-cell level .

How should ChIP-seq experiments be optimized for H4K12ac studies?

Optimizing ChIP-seq experiments for H4K12ac studies requires careful consideration of several parameters:

• Cell fixation conditions: Based on the provided search results, optimal fixation occurs with 1% formaldehyde for 10 minutes, which preserves protein-DNA interactions while maintaining antibody accessibility to the H4K12ac epitope .

• Cell quantity and antibody amount: For reliable results, using approximately 10^7 cells with 4 μg of anti-H4K12ac antibody provides sufficient material for high-quality ChIP-seq data .

• Sequencing depth: A minimum sequencing depth of 60 million reads is recommended for comprehensive genome-wide profiling of H4K12ac, as demonstrated by successful experiments on the Illumina NovaSeq 6000 platform .

• Control samples: Always include input control (non-immunoprecipitated chromatin) to normalize for biases in chromatin preparation and sequencing .

• Antibody validation: Verify antibody specificity using peptide competition assays or peptide array analysis prior to ChIP-seq experiments to ensure specific enrichment of H4K12ac-associated regions .

What are the critical factors affecting H4K12ac antibody specificity?

The specificity of H4K12ac antibodies is influenced by several factors that researchers must carefully consider:

• Cross-reactivity with similar modifications: H4K12ac antibodies may potentially cross-react with acetylation at other lysine residues on histone H4 or even other histones. Peptide array analysis, as shown in the search results, is critical for determining antibody specificity across a spectrum of histone modifications .

• Antibody format and clone: Different antibody formats (monoclonal vs. polyclonal) demonstrate varying specificity profiles. Monoclonal antibodies like EPR28340-173 show higher specificity than polyclonal alternatives in many applications .

• Adjacent modifications: The recognition of H4K12ac can be affected by modifications at adjacent amino acid residues, creating "epitope occlusion" where one modification prevents antibody binding to another nearby modification.

• Validation methods: Proper validation through peptide competition assays demonstrates specificity, as seen in immunohistochemistry results where signal disappears in the presence of competing acetylated peptide .

How can false negatives in H4K12ac detection be prevented?

False negatives in H4K12ac detection can result from several experimental factors:

• Histone deacetylase activity: Endogenous histone deacetylases can remove acetyl groups during sample preparation. Using histone deacetylase inhibitors like Trichostatin A (TSA) at 500 ng/mL for 4 hours can significantly enhance H4K12ac signal detection, as demonstrated in immunofluorescence and flow cytometry experiments .

• Fixation conditions: Excessive fixation can mask epitopes, while insufficient fixation may not preserve modifications. The optimal condition of 4% paraformaldehyde followed by permeabilization with 0.1% Triton X-100 has been validated for immunofluorescence applications .

• Antibody dilution: Using the appropriate antibody concentration is crucial for specific signal detection. For the rabbit monoclonal antibody [EPR28340-173], dilutions of 1/500 (1.044 μg/ml) for immunofluorescence, 1/50 for flow cytometry, and 1/1000 for western blot have been validated .

• Detection systems: Using high-sensitivity detection systems such as Goat Anti-Rabbit IgG H&L (Alexa Fluor® 488) at 1/1000 (2 μg/mL) dilution for immunofluorescence optimizes signal detection .

What controls should be included when performing H4K12ac immunoprecipitation experiments?

When performing H4K12ac immunoprecipitation experiments, the following controls are essential:

• Isotype control: Use of an isotype-matched irrelevant antibody (e.g., Rabbit IgG monoclonal [EPR25A]) to assess non-specific binding. The search results demonstrate this control in HeLa cell IP experiments .

• Input control: A small portion of the pre-immunoprecipitated material should be set aside to determine enrichment following immunoprecipitation .

• Positive and negative treatment controls: Treatment with histone deacetylase inhibitors (e.g., TSA at 500 ng/mL for 4 hours) serves as a positive control by increasing acetylation levels, while untreated cells provide a baseline for comparison .

• Peptide competition: Pre-incubation of the antibody with the acetylated peptide antigen should abolish specific signal, confirming antibody specificity .

• Secondary antibody-only control: Incubating samples with secondary antibody alone helps identify non-specific binding of the secondary antibody .

How can H4K12ac patterns be correlated with transcriptional activity?

Correlating H4K12ac patterns with transcriptional activity requires integrative analysis approaches:

• ChIP-seq and RNA-seq integration: Combining H4K12ac ChIP-seq data with RNA-seq from the same cells enables direct correlation between H4K12ac enrichment and gene expression levels. The optimized ChIP-seq protocol using 10^7 cells and 4 μg antibody followed by deep sequencing provides high-quality data for such integration .

• Genome browser visualization: Alignment of H4K12ac ChIP-seq tracks with RNA-seq and other epigenetic marks in genome browsers facilitates identification of correlations at specific genomic loci.

• Perturbation studies: Comparing H4K12ac distribution before and after treatment with compounds that alter histone acetylation (like TSA) alongside changes in gene expression provides functional insights into the role of this modification .

• Quantitative analysis: Calculating enrichment of H4K12ac at promoters, enhancers, and gene bodies followed by correlation with expression levels of associated genes can reveal distinct regulatory relationships.

What are effective approaches for multiplexing H4K12ac with other histone modifications?

Effective multiplexing of H4K12ac with other histone modifications can be achieved through several approaches:

• Sequential ChIP: Performing consecutive immunoprecipitations allows detection of co-occurrence of different modifications on the same nucleosomes.

• Multi-color immunofluorescence: The search results demonstrate successful immunofluorescence detection of H4K12ac (using Alexa Fluor® 488) alongside alpha-tubulin (using Alexa Fluor® 594) and nuclear DNA (DAPI), showing that multiple targets can be visualized simultaneously .

• Mass cytometry (CyTOF): The conjugation-ready antibody format mentioned in the search results is compatible with metal isotope labeling for mass cytometry, enabling simultaneous detection of numerous histone modifications at the single-cell level .

• Multiplex imaging: Advanced imaging techniques using conjugation-ready antibodies labeled with different fluorochromes or other detection systems allow visualization of multiple histone modifications in the same sample .

How does H4K12ac distribution differ across cell types and disease states?

The distribution of H4K12ac can vary significantly across different cellular contexts:

• Cell type variations: While the search results primarily focus on HeLa and COS7 cell lines, they indicate that H4K12ac patterns can be cell-type specific. The antibodies have been validated for detection of H4K12ac in human and other mammalian cells, including mouse and African green monkey samples .

• Disease associations: Altered H4K12ac patterns are observed in various disease contexts. The search results include immunohistochemistry analysis of human breast carcinoma tissue using H4K12ac antibodies, suggesting applications in cancer research .

• Response to treatment: Treatment with histone deacetylase inhibitors such as Trichostatin A (TSA) at 400-500 ng/mL for 4-24 hours consistently increases H4K12ac levels across different cell types, including HeLa, COS7, and HEK-293 cells .

What are the recommended approaches for studying H4K12ac in fixed tissue samples?

Studying H4K12ac in fixed tissue samples requires specific methodological considerations:

• Tissue fixation and processing: Paraffin-embedded tissue sections have been successfully used for H4K12ac detection, as demonstrated in human breast carcinoma tissue analysis .

• Antibody dilution optimization: For immunohistochemistry applications, dilutions between 1/50 - 1/100 of the polyclonal H4K12ac antibody have been validated .

• Antigen retrieval: Although not explicitly mentioned in the search results, antigen retrieval is typically necessary for optimal detection of histone modifications in fixed tissue samples.

• Validation with peptide competition: Including controls with competing acetylated peptide is crucial for confirming specificity in tissue samples, as demonstrated in the immunohistochemistry results for human breast carcinoma tissue .

• Counterstaining: Appropriate counterstains should be selected to provide context for H4K12ac localization within tissue architecture without interfering with the primary signal.

What are the key differences between monoclonal and polyclonal H4K12ac antibodies?

Understanding the differences between antibody types is crucial for experimental design:

• Specificity profiles: The search results indicate that rabbit monoclonal antibodies like EPR28340-173 demonstrate high specificity for H4K12ac as confirmed by peptide array analysis against 501 different modified and unmodified histone peptides . Polyclonal antibodies may recognize multiple epitopes but require careful validation .

• Applications compatibility: Both types show utility across multiple applications, but the monoclonal antibody [EPR28340-173] has been validated for more advanced applications including ChIP-seq, while polyclonal alternatives are primarily validated for standard applications like western blot, immunohistochemistry, and immunofluorescence .

• Reproducibility: Monoclonal antibodies generally offer greater lot-to-lot consistency, which is particularly important for longitudinal studies or comparison between datasets generated at different times.

• Sensitivity in different applications: The rabbit monoclonal antibody demonstrates excellent sensitivity in ChIP-seq and flow cytometry applications with clear enrichment of H4K12ac signal in TSA-treated cells compared to untreated controls .

How can peptide array analysis inform antibody selection for specific experiments?

Peptide array analysis provides crucial information for antibody selection:

• Cross-reactivity assessment: The search results mention peptide array analysis against 501 different modified and unmodified histone peptides, which can identify potential cross-reactivity with similar histone modifications .

• Epitope occlusion detection: Peptide arrays can reveal whether modifications near the target lysine affect antibody binding, informing experimental design especially when studying co-occurring modifications.

• Quantitative affinity comparisons: The search results describe calculating affinity as the area under the curve when antibody binding values are plotted against corresponding peptide concentration, with each circle area normalized to the peptide with strongest affinity .

• Application-specific selection: For applications requiring the highest specificity (e.g., ChIP-seq), selecting antibodies with minimal cross-reactivity in peptide array analysis is essential, while some cross-reactivity might be tolerable for applications like western blotting where size separation provides additional specificity.