β-hydroxybutyryl-HIST1H3A (K18) Antibody

What is histone lysine β-hydroxybutyrylation and how does it differ from other histone modifications?

Histone lysine β-hydroxybutyrylation (Kbhb) is a novel post-translational modification (PTM) derived from β-hydroxybutyrate (BHB), a primary ketone body. Unlike better-characterized modifications such as acetylation or methylation, Kbhb specifically responds to cellular metabolic states, particularly during carbohydrate restriction or elevated BHB levels. Structurally, β-hydroxybutyrylation involves the addition of a β-hydroxybutyryl group (containing a hydroxyl group at the beta position) to lysine residues on histones, creating a unique molecular signature with distinct regulatory potential .

While acetylation primarily neutralizes the positive charge of lysine residues to alter chromatin structure, β-hydroxybutyrylation introduces both charge neutralization and additional structural properties through its hydroxyl group. These distinctive biochemical characteristics may explain why Kbhb demonstrates different regulatory functions from histone acetylation and methylation, particularly in metabolic stress responses . To experimentally distinguish between these modifications, researchers must employ modification-specific antibodies and/or mass spectrometry-based proteomics approaches.

What are the key sites of histone β-hydroxybutyrylation identified to date?

Current research has identified 44 histone Kbhb sites across the proteome, a number comparable to known histone acetylation sites . The most extensively characterized modification sites include:

H3K18bhb serves as a particularly important site in metabolic regulation, with documented roles in starvation-responsive gene expression . Studies have shown that H3K9bhb, H3K18bhb, H4K8bhb, and H3K4bhb levels increase in a β-hydroxybutyrate dose-dependent manner, suggesting site-specific sensitivity to metabolic fluctuations . Researchers investigating metabolic signaling through chromatin should prioritize these sites in their experimental designs.

How should researchers optimize chromatin immunoprecipitation (ChIP) protocols when using β-hydroxybutyryl-HIST1H3A (K18) antibodies?

For optimal ChIP results with β-hydroxybutyryl-HIST1H3A (K18) antibodies, researchers should implement the following methodological considerations:

• Cross-linking optimization: Use 1% formaldehyde for 10 minutes at room temperature, as excessive cross-linking can mask the β-hydroxybutyrylated epitope.

• Sonication parameters: Adjust sonication conditions to generate chromatin fragments of 200-500 bp, which is optimal for histone modification ChIP.

• Antibody concentration: For β-hydroxybutyryl-HIST1H3A (K18) antibodies, use a 1:50 dilution for ChIP applications, which typically corresponds to 2-4 μg of antibody per ChIP reaction .

• Validation controls: Always include:

  • Input control (non-immunoprecipitated chromatin)

  • IgG negative control

  • Positive control targeting abundant histone marks (e.g., H3K4me3)

  • Spike-in controls for quantitative analyses

• Washing stringency: For β-hydroxybutyrylation ChIP, include an additional high-salt wash (500 mM NaCl) to reduce background without compromising specific signal.

When performing subsequent qPCR analysis, design primers for genomic regions known to be enriched in H3K18bhb marks, particularly starvation-responsive gene promoters, to serve as positive controls .

What are the recommended protocols for immunofluorescence (IF) staining with β-hydroxybutyryl-HIST1H3A (K18) antibody?

For effective immunofluorescence detection of β-hydroxybutyryl-HIST1H3A (K18), researchers should follow this optimized protocol:

• Cell preparation: Fix cells with 4% paraformaldehyde for 15 minutes at room temperature, followed by permeabilization with 0.2% Triton X-100 for 10 minutes.

• Antigen retrieval: Perform mild antigen retrieval using 10 mM sodium citrate buffer (pH 6.0) at 95°C for 10 minutes to enhance epitope accessibility.

• Blocking: Block with 5% BSA in PBS for 1 hour at room temperature to minimize non-specific binding.

• Primary antibody incubation: Dilute β-hydroxybutyryl-HIST1H3A (K18) antibody at 1:20-1:200 in blocking buffer and incubate overnight at 4°C .

• Secondary antibody: Use appropriate fluorophore-conjugated secondary antibodies (anti-rabbit IgG) at 1:500 dilution for 1 hour at room temperature.

• Nuclear counterstaining: Counterstain with DAPI (1 μg/ml) for 5 minutes.

• Controls: Include parallel samples with:

  • Primary antibody omission

  • Peptide competition (using the immunizing peptide)

  • Dual staining with other histone marks for colocalization studies

For quantitative IF analysis, acquire images using identical exposure settings and analyze signal intensity within the nuclear compartment using appropriate image analysis software.

How can researchers effectively distinguish between β-hydroxybutyrylation and other acyl modifications in histone analysis?

Distinguishing between β-hydroxybutyrylation and other acyl modifications requires a multi-faceted analytical approach:

• Antibody specificity validation: Verify antibody specificity through:

  • Dot blot assays with modified and unmodified peptides

  • Competition experiments with structurally related modified peptides

  • Western blot analysis following HDAC treatment

• Mass spectrometry differentiation:

  • Use targeted MS/MS approaches with specific diagnostic fragment ions

  • Employ high-resolution MS to distinguish between isobaric modifications

  • Implement stable isotope labeling with isotopic β-hydroxybutyrate (e.g., [13C2]) to definitively identify β-hydroxybutyrylated peptides through mass shift detection

• Chiral analysis: Consider the chirality of β-hydroxybutyrylation, as R-β-hydroxybutyrylation and S-β-hydroxybutyrylation exhibit different interactions with deacetylases like HDAC3 and SIRT3 .

• Enzymatic specificity tests: Utilize the differential sensitivity of modifications to specific erasers (HDACs and SIRTs) as HDAC1-3 and SIRT1-3 have been identified to have de-β-hydroxybutyrylation activity, though with varying specificities .

This comprehensive approach enables researchers to definitively identify β-hydroxybutyrylation against the complex background of cellular acylation patterns, avoiding misattribution of biological functions.

What are the most effective approaches for studying the dynamic interplay between histone β-hydroxybutyrylation and metabolic regulation?

To investigate the dynamic relationship between histone β-hydroxybutyrylation and metabolic signaling, researchers should implement these advanced approaches:

• Metabolic manipulation models:

  • Fasting/feeding cycles (16-24h fasting periods)

  • Ketogenic diet administration

  • Streptozotocin-induced diabetic ketoacidosis

  • Direct β-hydroxybutyrate supplementation (5-10 mM for cell culture)

• Integrated multi-omics:

  • Parallel ChIP-seq of H3K18bhb and other histone marks

  • RNA-seq to correlate histone modifications with transcriptional outputs

  • Metabolomics to measure BHB and related metabolite levels

  • Proteomics to assess global protein β-hydroxybutyrylation

• Temporal resolution studies:

  • Time-course experiments following metabolic perturbation

  • Pulse-chase labeling with isotopic BHB to determine modification turnover rates

• Enzymatic manipulation:

  • p300 knockdown/inhibition to disrupt the "writing" of Kbhb marks

  • HDAC1-3 or SIRT1-3 modulation to alter "erasing" activities

  • BHB-CoA synthetase manipulation to control BHB activation

Such integrated approaches allow researchers to establish causal relationships between metabolic states, histone β-hydroxybutyrylation patterns, and downstream gene expression changes, particularly in starvation-responsive metabolic pathways where H3K18bhb has demonstrated regulatory roles .

What are common issues encountered in Western blotting with β-hydroxybutyryl-HIST1H3A (K18) antibodies and how can they be resolved?

For optimal detection, researchers should:

• Use fresh histones extracted with acidic extraction methods

• Run 15-18% SDS-PAGE gels for better resolution of histone proteins

• Transfer to PVDF membranes (rather than nitrocellulose) at 30V overnight at 4°C

• Use recommended dilutions (1:100-1:1000) for Western blot applications

• Include parallel blots for total H3 as loading controls

How can researchers accurately quantify changes in histone β-hydroxybutyrylation levels under different experimental conditions?

For accurate quantification of histone β-hydroxybutyrylation changes, researchers should implement these methodological approaches:

• Western blot quantification:

  • Use infrared fluorescence-based detection systems rather than chemiluminescence

  • Normalize H3K18bhb signal to total H3 signal from the same membrane (after stripping) or parallel membrane

  • Include standard curves with known quantities of modified peptides

  • Apply appropriate statistical analyses (minimum of three biological replicates)

• Mass spectrometry-based quantification:

  • Implement label-free quantification of modified peptides with appropriate normalization

  • Use SILAC or TMT labeling for more accurate comparative analysis

  • Monitor multiple β-hydroxybutyrylated peptides, not just H3K18bhb

  • Calculate modification stoichiometry (percentage of a site carrying the modification)

• ChIP-seq quantification:

  • Use spike-in normalization with exogenous chromatin

  • Apply appropriate normalization methods for between-sample comparisons

  • Calculate differential binding metrics rather than raw peaks

  • Correlate with gene expression changes for functional significance

• Experimental design considerations:

  • Include time-course measurements to capture dynamic changes

  • Use appropriate metabolic controls (e.g., acetate treatment, glucose manipulation)

  • Include both technical and biological replicates

  • Implement multiple independent methods for cross-validation

Proper quantification is essential as histone β-hydroxybutyrylation levels change in response to β-hydroxybutyrate concentration in a dose-dependent manner, with H3K9bhb, H3K18bhb, H4K8bhb, and H3K4bhb all showing differential sensitivity to metabolic fluctuations .

How might researchers investigate the relationship between histone β-hydroxybutyrylation and disease pathogenesis?

To explore the connections between histone β-hydroxybutyrylation and disease states, researchers should consider these advanced investigative approaches:

• Clinical sample analysis:

  • Compare β-hydroxybutyrylation patterns in patient-derived tissues and matched controls

  • Correlate H3K18bhb levels with clinical parameters and disease progression

  • Develop tissue microarray analyses with β-hydroxybutyrylation antibodies

• Disease model integration:

  • Examine β-hydroxybutyrylation in established disease models of metabolic disorders, cardiovascular diseases, kidney diseases, and cancer

  • Create genetic models with altered β-hydroxybutyrylation machinery (p300 mutants, HDAC mutants)

  • Implement diet and pharmacological interventions that modulate BHB levels

• Therapeutic targeting strategies:

  • Screen for specific inhibitors/activators of histone β-hydroxybutyrylation

  • Investigate BHB precursors as potential epigenetic therapeutics

  • Explore combination approaches targeting multiple epigenetic marks

• Mechanistic investigations:

  • Identify disease-specific β-hydroxybutyrylation regulatory networks

  • Determine the interplay between β-hydroxybutyrylation and inflammation pathways

  • Explore β-hydroxybutyrylation in cellular stress responses relevant to disease

This research is particularly promising given that histone Kbhb has been associated with the pathogenesis of metabolic cardiovascular diseases, kidney diseases, tumors, neuropsychiatric disorders, and metabolic diseases with functions distinct from histone acetylation and methylation .

What experimental approaches can reveal the writers and erasers specific to histone β-hydroxybutyrylation?

To identify and characterize the enzymatic machinery regulating histone β-hydroxybutyrylation, researchers should implement these specialized approaches:

• Enzyme activity profiling:

  • In vitro assays with recombinant enzymes and isotope-labeled BHB-CoA

  • Test known acyltransferases (particularly p300) for β-hydroxybutyrylation activity

  • Screen HDAC and SIRT family members for de-β-hydroxybutyrylation activity

  • Investigate enzyme kinetics and substrate preferences

• Protein-protein interaction studies:

  • Perform BioID or proximity labeling with potential writer/eraser enzymes

  • Use co-immunoprecipitation to identify protein complexes involved in β-hydroxybutyrylation

  • Implement FRET-based approaches to detect dynamic enzyme-substrate interactions

• Genetic screening:

  • Conduct CRISPR screens targeting epigenetic regulators and assess β-hydroxybutyrylation levels

  • Generate knockout/knockdown models of candidate enzymes

  • Perform rescue experiments with wild-type and catalytically dead enzyme variants

• Structural biology approaches:

  • Determine crystal structures of enzymes in complex with β-hydroxybutyrylated peptides

  • Investigate binding pockets and catalytic mechanisms

  • Explore the structural basis for chiral preferences (R- vs. S-β-hydroxybutyrylation)

Current research has identified p300 as a histone Kbhb "writer" that catalyzes the addition of BHB to lysine residues, with particular activity at H3K9, H3K18, H3K27, and H4K8 sites . For erasers, HDAC1-3 and SIRT1-3 have demonstrated de-β-hydroxybutyrylation activity in vitro, with HDAC1 and HDAC2 functioning as primary deacylases in cellular contexts . The differential activity of these enzymes on R- versus S-β-hydroxybutyrylation highlights the complexity of this regulatory system and presents opportunities for targeted manipulation.

What are the critical factors for successful ChIP-seq experiments using β-hydroxybutyryl-HIST1H3A (K18) antibodies?

For high-quality ChIP-seq data with β-hydroxybutyryl-HIST1H3A (K18) antibodies, researchers should address these critical experimental parameters:

• Antibody validation for ChIP-seq:

  • Confirm antibody specificity via Western blot and peptide competition

  • Perform preliminary ChIP-qPCR at known target regions before sequencing

  • Validate antibody lot-to-lot consistency with standard samples

• Chromatin preparation optimization:

  • Use dual cross-linking (formaldehyde followed by EGS) for enhanced capture

  • Optimize sonication for consistent fragment size distribution (200-400 bp)

  • Implement stringent quality control of chromatin (fragment analysis, DNA concentration)

• Library preparation considerations:

  • Use ChIP-seq optimized library preparation kits with minimal PCR cycles

  • Include appropriate controls (input, IgG, spike-in)

  • Implement unique molecular identifiers (UMIs) to control for PCR duplication

• Bioinformatic analysis pipeline:

  • Use peak callers optimized for histone modifications (e.g., MACS2 with broad peak settings)

  • Implement robust normalization methods

  • Perform integrative analysis with RNA-seq and other epigenomic data

  • Consider differential binding analysis rather than binary peak calling

ChIP-seq studies have demonstrated that histone Kbhb is enriched in active gene promoters, particularly those associated with starvation-responsive metabolic pathways . When designing ChIP-seq experiments, researchers should consider metabolic perturbations that elevate cellular β-hydroxybutyrate levels to enhance the detection of differential binding patterns.

How can researchers effectively use β-hydroxybutyryl-HIST1H3A (K18) antibodies in multi-parameter flow cytometry or CyTOF applications?

For multiplexed flow cytometry or mass cytometry (CyTOF) applications with β-hydroxybutyryl-HIST1H3A (K18) antibodies, implement these specialized protocols:

• Sample preparation optimization:

  • Use gentle fixation (1% formaldehyde, 10 minutes) followed by methanol permeabilization

  • Implement specialized nuclear preparation kits for consistent results

  • Include RNase treatment to reduce background

• Antibody labeling strategies:

  • For flow cytometry: Use bright fluorophores (Alexa Fluor 488 or PE) for histone modifications

  • For CyTOF: Label with rare earth metals with minimal signal overlap

  • Titrate antibody concentrations carefully to optimize signal-to-noise ratio

• Panel design considerations:

  • Include markers for cell cycle phases (e.g., Ki-67, PCNA)

  • Add other histone modifications for correlation analysis

  • Include metabolic state markers when possible

• Analysis approaches:

  • Implement dimensionality reduction techniques (tSNE, UMAP)

  • Use advanced clustering algorithms to identify cell populations with distinct modification patterns

  • Consider trajectory analysis for dynamic processes

• Validation requirements:

  • Confirm patterns with imaging flow cytometry

  • Validate with orthogonal techniques (Western blot, ChIP)

  • Include appropriate biological controls (metabolic perturbations)

This approach enables single-cell resolution analysis of β-hydroxybutyrylation dynamics and heterogeneity within complex populations, revealing cell state-specific regulation that may be obscured in bulk analyses.