β-hydroxybutyryl-HIST1H3A (K23) Antibody

What is the biological significance of histone β-hydroxybutyrylation at K23?

Histone H3 lysine 23 β-hydroxybutyrylation (H3K23bhb) represents an evolutionarily conserved post-translational modification (PTM) that plays a critical role in chromatin regulation. Like other histone modifications, H3K23bhb affects chromatin structure by altering DNA accessibility to cellular machinery that requires DNA as a template . This modification is part of the complex "histone code" that regulates transcription, DNA repair, DNA replication, and chromosomal stability . Research has demonstrated that β-hydroxybutyrylation is widespread across histone proteins, with at least 44 identified sites across multiple histone variants . The K23 position on histone H3 is particularly significant as its modification status can directly influence gene expression patterns through recruitment of specific reader proteins and modulation of chromatin compaction.

What are the recommended applications for β-hydroxybutyryl-HIST1H3A (K23) antibody?

The β-hydroxybutyryl-HIST1H3A (K23) antibody is validated for multiple research applications that enable comprehensive investigation of this histone modification. Primary recommended applications include Enzyme-Linked Immunosorbent Assay (ELISA), Western Blotting (WB), and Chromatin Immunoprecipitation (ChIP) . For Western blotting applications, the recommended dilution range is 1:100-1:1000, which should be optimized for specific experimental conditions . The antibody has been confirmed to detect endogenous levels of β-hydroxybutyryl-HIST1H3A (K23) protein with high specificity . Additionally, this antibody can be utilized in chromatin immunoprecipitation coupled with sequencing (ChIP-seq) to map the genomic distribution of H3K23bhb marks, providing insights into their role in gene regulation . When conducting immunofluorescence studies, this antibody enables visualization of the nuclear localization pattern of β-hydroxybutyrylated histones .

How should researchers validate the specificity of β-hydroxybutyryl-HIST1H3A (K23) antibody?

Rigorous validation of antibody specificity is essential for accurate interpretation of histone modification studies. A comprehensive validation protocol should include multiple complementary approaches. First, peptide competition assays can determine whether the antibody binding is specifically inhibited by the target modified peptide but not by unmodified or differently modified peptides . Second, peptide microarray analysis provides a high-throughput method to assess cross-reactivity against various histone modifications . The ArrayNinja software package facilitates the design, fabrication, and analysis of such microarrays for antibody specificity profiling . Third, dot blot assays using synthetic peptides with different modifications can quickly screen for specificity, as demonstrated for pan anti-Kbhb antibodies . Fourth, immunoblotting should show the expected molecular weight band (approximately 16 KDa for histone H3) and appropriate response to treatments that alter β-hydroxybutyrylation levels, such as sodium β-hydroxybutyrate exposure . Finally, mass spectrometry validation of immunoprecipitated histones provides the most definitive confirmation of antibody specificity.

What storage conditions maximize the stability and activity of β-hydroxybutyryl-HIST1H3A (K23) antibody?

Proper storage is critical for maintaining antibody function and preventing activity loss. For short-term storage (up to one week), store the β-hydroxybutyryl-HIST1H3A (K23) antibody at +4°C . For long-term storage, aliquot the antibody and maintain at -20°C or preferably -80°C to prevent degradation . It is crucial to avoid repeated freeze-thaw cycles, as each cycle can result in approximately 50% loss of binding activity . Small aliquots appropriate for individual experiments should be prepared upon receipt of the antibody. When working with the antibody, keep it on ice and minimize exposure to room temperature. If any precipitation occurs, centrifuge the antibody solution before use. For diluted working solutions, prepare them fresh before each experiment and do not store diluted antibody for extended periods. Adding preservatives such as sodium azide (0.02%) can help prevent microbial contamination during storage, but ensure this doesn't interfere with downstream applications.

What controls should be included when performing ChIP experiments with β-hydroxybutyryl-HIST1H3A (K23) antibody?

Chromatin immunoprecipitation experiments require rigorous controls to ensure reliable interpretation of results. For ChIP experiments with β-hydroxybutyryl-HIST1H3A (K23) antibody, implement the following control strategy:

Additionally, when comparing β-hydroxybutyrylation with other modifications like acetylation, parallel ChIP experiments should be performed with antibodies against H3K23ac and total H3, allowing normalization and comparative analysis of different histone marks at the same genomic locations .

How can metabolic state influence β-hydroxybutyrylation at H3K23, and how might this be experimentally manipulated?

Cellular metabolic state directly impacts histone β-hydroxybutyrylation levels through modulation of β-hydroxybutyrate (bhb) availability and β-hydroxybutyryl-CoA production. Research has demonstrated that β-hydroxybutyryl-CoA serves as the cofactor for lysine β-hydroxybutyrylation, with isotopic tracing experiments confirming that exogenous sodium β-hydroxybutyrate can be converted to bhb-CoA in cells . To experimentally manipulate H3K23bhb levels, researchers can employ several metabolic intervention strategies:

• Direct supplementation with sodium β-hydroxybutyrate (NaBHB) at concentrations of 5-10 mM induces dose-dependent increases in histone Kbhb levels .

• Fasting conditions elevate endogenous β-hydroxybutyrate production, resulting in increased hepatic histone Kbhb levels in mouse models .

• Diabetic conditions, such as those induced by streptozotocin (STZ) treatment in mice, lead to elevated β-hydroxybutyrate production and corresponding increases in histone Kbhb .

• Ketogenic diets, which promote hepatic ketogenesis and β-hydroxybutyrate production, can be used to elevate histone Kbhb levels in vivo.

When conducting such metabolic manipulation experiments, researchers should monitor cellular β-hydroxybutyrate concentrations alongside histone modifications to establish dose-response relationships. Comparative analysis with other histone modifications (particularly acetylation) is recommended to distinguish modification-specific effects from global chromatin changes.

What approaches can be used to distinguish between different histone modifications at the K23 position in multiplexed experiments?

Distinguishing between different modifications at the same histone residue requires sophisticated analytical approaches to prevent cross-reactivity and ensure accurate identification. For the H3K23 position, which can undergo multiple modifications including β-hydroxybutyrylation and acetylation, researchers should employ a multi-modal strategy:

• Sequential chromatin immunoprecipitation (Re-ChIP) can determine whether different modifications co-occur on the same histone tail by performing successive immunoprecipitations with antibodies against different modifications (e.g., first with anti-H3K23bhb, then with anti-H3K23ac).

• Peptide competition assays with modified peptide arrays can test antibody specificity against a panel of differently modified peptides to ensure the anti-H3K23bhb antibody does not cross-react with H3K23ac or other modifications .

• Mass spectrometry-based approaches, particularly bottom-up proteomics of histone peptides, provide the most definitive method for identifying and quantifying specific modifications. This approach can determine the relative abundance of each modification type at K23.

• Multiplexed immunofluorescence using carefully validated antibodies with distinct fluorophores can visualize the distribution patterns of different modifications within the nucleus.

• For genomic studies, parallel ChIP-seq experiments with modification-specific antibodies followed by integrated computational analysis can reveal distinct or overlapping distribution patterns.

These approaches, when used in combination, enable robust discrimination between β-hydroxybutyrylation and other modifications at the H3K23 position.

What is the relationship between β-hydroxybutyrylation and other histone marks in the context of gene regulation?

Histone β-hydroxybutyrylation functions within a complex ecosystem of post-translational modifications that collectively regulate gene expression. Understanding the interplay between H3K23bhb and other histone marks provides insights into the functional outcomes of this modification. Research has identified several key relationships:

• Comparative analysis of histone modifications has revealed 44 distinct histone Kbhb sites, many of which occur at lysine residues where acetylation and methylation are also known to play important roles in chromatin regulation (including H4K8, H4K12, H3K4, H3K9, and H3K56) .

• While β-hydroxybutyrylation and acetylation can occur at the same lysine residues, they appear to be independently regulated. When cells are treated with β-hydroxybutyrate, Kbhb levels increase significantly while acetylation levels remain largely unchanged .

• The genomic distribution of different histone marks provides functional insights. ChIP-seq analysis of histone modifications has revealed that specific patterns of co-occurrence or mutual exclusion with other marks can indicate functional genomic elements (enhancers, promoters, etc.) and transcriptional states.

• The dynamics of β-hydroxybutyrylation during metabolic changes (fasting, diabetes) suggest this modification may serve as a mechanism for coupling cellular metabolism to gene expression programs, potentially through recruitment of specific reader proteins that recognize this modification .

To fully characterize these relationships, researchers should employ integrated multi-omics approaches combining ChIP-seq for multiple histone marks with transcriptome analysis (RNA-seq) to correlate modification patterns with gene expression changes under various metabolic conditions.

What are common issues in Western blot experiments with β-hydroxybutyryl-HIST1H3A (K23) antibody and how can they be resolved?

Western blotting with histone modification antibodies presents several technical challenges that require specific optimization strategies. For β-hydroxybutyryl-HIST1H3A (K23) antibody, researchers may encounter the following issues:

Additionally, when working with histone modifications that are sensitive to metabolic state, such as β-hydroxybutyrylation, it is crucial to standardize cell culture conditions and harvesting protocols to minimize variation in cellular metabolite levels. Pre-treatment with sodium β-hydroxybutyrate (5-10 mM) can serve as a positive control to enhance H3K23bhb signal .

How can ChIP-seq experiments with β-hydroxybutyryl-HIST1H3A (K23) antibody be optimized for maximum sensitivity and specificity?

Optimizing ChIP-seq experiments for β-hydroxybutyryl-HIST1H3A (K23) requires careful consideration of several parameters to ensure high signal-to-noise ratio and reproducible results:

• Crosslinking optimization: Standard 1% formaldehyde for 10 minutes may be suitable, but optimization for histone modifications is recommended. Test different crosslinking times (5-15 minutes) to maximize signal while minimizing epitope masking.

• Sonication parameters: Aim for chromatin fragments of 200-500 bp. Over-sonication can damage epitopes while under-sonication reduces resolution. Verify fragment size by agarose gel electrophoresis.

• Antibody amount: Titrate antibody concentration to determine the optimal amount that maximizes specific signal while minimizing background. For β-hydroxybutyryl-HIST1H3A (K23) antibody, begin with manufacturer recommendations and adjust based on results.

• Washing stringency: Balance between preserving specific interactions and reducing background. Include at least one high-salt wash (500 mM NaCl) to reduce non-specific binding.

• Input normalization: Always sequence an input control from the same chromatin preparation to correct for biases in chromatin accessibility and sequencing.

• Spike-in normalization: Consider using spike-in controls (e.g., Drosophila chromatin with Drosophila-specific antibody) for quantitative comparisons across conditions, particularly when global levels of β-hydroxybutyrylation may change.

• Metabolic manipulation: When comparing β-hydroxybutyrylation patterns across different metabolic states, include sodium β-hydroxybutyrate-treated samples (10 mM) as positive controls .

• Sequencing depth: For histone modifications with potentially broad distribution patterns like β-hydroxybutyrylation, aim for at least 20 million uniquely mapped reads per sample to ensure comprehensive coverage.

By systematically optimizing these parameters, researchers can generate high-quality ChIP-seq data for β-hydroxybutyryl-HIST1H3A (K23) that enables reliable identification of genomic regions enriched for this modification.

What are emerging techniques for studying β-hydroxybutyrylation dynamics in live cells?

Understanding the dynamic nature of histone β-hydroxybutyrylation in response to changing metabolic conditions requires advanced techniques for real-time monitoring in living cells. Several emerging approaches show promise for future research:

• FRET-based sensors: Developing Förster resonance energy transfer (FRET) sensors consisting of β-hydroxybutyryl-specific reader domains coupled to fluorescent protein pairs could enable real-time visualization of β-hydroxybutyrylation changes in living cells.

• Click chemistry approaches: Metabolic labeling with alkyne or azide-modified β-hydroxybutyrate analogs, followed by bioorthogonal click chemistry reactions, can facilitate pulse-chase experiments to track the kinetics of β-hydroxybutyrylation turnover.

• Engineered reader domains: Modified versions of natural β-hydroxybutyrylation reader proteins, fused to fluorescent markers, can be expressed in cells to track the dynamics and nuclear localization patterns of β-hydroxybutyrylated histones.

• CRISPR-based epigenetic editing: dCas9 fused to enzymes that catalyze or remove β-hydroxybutyrylation can be used to manipulate this modification at specific genomic loci and monitor consequent functional effects.

• Single-molecule imaging: Advanced microscopy techniques combined with site-specific incorporation of fluorescent β-hydroxybutyryl analogs could potentially track individual modification events at the single-molecule level.

These emerging techniques, when developed and validated, will complement existing antibody-based approaches to provide a more comprehensive understanding of β-hydroxybutyrylation dynamics in cellular contexts.

How might disruptions in histone β-hydroxybutyrylation contribute to metabolic disease pathogenesis?

The connection between histone β-hydroxybutyrylation and cellular metabolism suggests potential implications for metabolic disease mechanisms. Several lines of evidence support this relationship:

• Studies have demonstrated elevated histone Kbhb levels in livers of both fasted and streptozotocin-induced diabetic mice, suggesting that altered β-hydroxybutyrylation patterns may be associated with metabolic dysregulation .

• As β-hydroxybutyrylation directly responds to cellular β-hydroxybutyrate levels, conditions characterized by ketosis (such as diabetic ketoacidosis, prolonged fasting, or ketogenic diets) likely feature altered histone β-hydroxybutyrylation patterns that could affect gene expression programs.

• The identification of 44 distinct histone Kbhb sites, many at functionally important lysine residues, suggests that β-hydroxybutyrylation may regulate genes involved in metabolic homeostasis .

• If β-hydroxybutyrylation and acetylation compete for the same lysine residues but respond differently to metabolic signals, disruption of this balance could potentially contribute to inappropriate gene expression patterns in metabolic diseases.

Future research directions should include comprehensive profiling of histone β-hydroxybutyrylation patterns in tissues from patients with various metabolic disorders, correlation of these patterns with gene expression changes, and mechanistic studies to determine whether alterations in β-hydroxybutyrylation are causative factors or consequences of disease states. Such research may identify novel therapeutic targets for metabolic diseases based on modulating specific histone modifications.