The β-hydroxybutyryl-HIST1H2AG (K36) Antibody is a polyclonal rabbit antibody designed to detect lysine β-hydroxybutyrylation at position 36 (K36) on histone H2A type 1 (HIST1H2AG). This post-translational modification (PTM) involves the covalent attachment of β-hydroxybutyrate (BHB), a ketone body, to lysine residues, linking metabolic states to chromatin regulation .
HIST1H2AG is a core histone protein critical for nucleosome assembly and chromatin structure. Its β-hydroxybutyrylation at K36 is part of a broader family of lysine β-hydroxybutyrylation (Kbhb) modifications, which are metabolically regulated and influence gene expression .
Synonym | Gene ID | Function |
---|---|---|
H2A.1 | HIST1H2AG | Chromatin compaction, transcriptional regulation |
H2AFP | DNA repair, replication, and chromosomal stability | |
H2AC11 | Histone code modification for epigenetic signaling |
β-hydroxybutyrylation is linked to cellular metabolism, particularly under conditions of fasting or ketosis. BHB, produced via β-oxidation, is converted to β-hydroxybutyryl-CoA, which serves as a cofactor for lysine β-hydroxybutyryltransferases .
Metabolic Regulation: Kbhb levels rise in response to elevated BHB, as seen in fasting, diabetes, or ketogenic states .
Gene Expression: Kbhb marks, including H2A K36, are enriched at active promoters and enhancers, promoting transcription of metabolic genes (e.g., mitochondrial oxidative phosphorylation pathways) .
Therapeutic Implications: In sarcopenia models, β-HB supplementation reverses muscle atrophy via H2A K36 β-hydroxybutyrylation, enhancing mitochondrial function .
The antibody is validated for detecting β-hydroxybutyryl-HIST1H2AG (K36) in human samples.
In HeLa cells treated with 30 mM sodium butyrate, the antibody detects a 15 kDa band corresponding to β-hydroxybutyrylated H2A .
While the β-hydroxybutyryl-HIST1H2AG (K36) Antibody is specific, cross-reactivity concerns exist for other Kbhb antibodies (e.g., H3K9bhb) .
Histone H2A (K36) is a core component of the nucleosome. Nucleosomes package and compact DNA into chromatin, thereby regulating DNA accessibility to cellular machinery requiring DNA as a template. Histones, therefore, play a crucial role in transcription regulation, DNA repair, DNA replication, and chromosomal stability. Control of DNA accessibility is achieved through a complex interplay of histone post-translational modifications, often referred to as the histone code, and nucleosome remodeling.
β-hydroxybutyryl-HIST1H2AG (K36) Antibody is a polyclonal antibody that specifically recognizes the β-hydroxybutyrylation post-translational modification at lysine 36 of Histone H2A type 1. This antibody has been generated using a peptide sequence surrounding the β-hydroxybutyryl-Lys (36) derived from Human Histone H2A type 1 as the immunogen . The antibody targets a specific histone mark that is part of the emerging landscape of histone lysine acylations, which play crucial roles in epigenetic regulation.
β-hydroxybutyrylation (Kbhb) is a relatively newly identified histone post-translational modification that differs from other acylations like acetylation in both structure and function. Unlike acetylation, β-hydroxybutyrylation is specifically induced by β-hydroxybutyrate (BHB), a ketone body that increases during fasting or diabetic ketoacidosis . While acetylation shows minimal changes during metabolic stress, histone Kbhb levels are dramatically elevated in response to increased β-hydroxybutyrate concentrations . Structurally, β-hydroxybutyrylation adds a larger, hydroxylated acyl group to the lysine residue, which may create distinct protein-protein interaction interfaces compared to acetylation or methylation.
The β-hydroxybutyryl-HIST1H2AG (K36) Polyclonal Antibody has been validated for enzyme-linked immunosorbent assay (ELISA) and Western blotting (WB) . These applications enable researchers to detect and quantify the presence of β-hydroxybutyrylated histones in various experimental settings, including cell culture systems and tissue samples from animal models. The antibody specificity allows for the monitoring of this modification under different physiological and pathological conditions.
To effectively study dynamic changes in histone β-hydroxybutyrylation, researchers should consider a multi-tiered experimental approach. First, establish baseline levels in your model system, then implement metabolic interventions known to affect β-hydroxybutyrate levels, such as:
Treating cultured cells with sodium β-hydroxybutyrate at various concentrations (e.g., 1-20 mM) for 24 hours
Subjecting animal models to fasting protocols (typically 24-48 hours for mice)
Utilizing diabetic models such as streptozotocin (STZ)-induced Type 1 diabetes in mice
Time-course experiments are crucial for capturing the dynamic nature of these modifications. Additionally, researchers should measure blood β-hydroxybutyrate levels concurrently with histone modifications to establish correlations. For comprehensive analysis, combine Western blotting with site-specific antibodies, mass spectrometry, and ChIP-seq to map genomic distribution patterns of the modification .
For optimal preservation of histone β-hydroxybutyrylation, implement these critical steps in your sample preparation protocol:
Harvest cells or tissues rapidly to minimize post-collection enzymatic activities
Include deacetylase inhibitors (e.g., trichostatin A) and β-hydroxybutyrylation deacylase inhibitors in lysis buffers
Perform histone extraction using acid extraction methods (typically with 0.2N HCl) followed by TCA precipitation
Store extracted histones at -80°C and avoid repeated freeze-thaw cycles
For tissue samples, flash-freezing in liquid nitrogen immediately after collection is essential. When preparing samples for Western blotting, use freshly prepared loading buffers and avoid excessive heating, which may affect the stability of the modification. For immunoprecipitation experiments, crosslinking conditions should be optimized to preserve the epitope recognized by the antibody .
To rigorously validate the specificity of β-hydroxybutyryl-HIST1H2AG (K36) Antibody in your experimental system, implement these approaches:
Peptide competition assay: Pre-incubate the antibody with excess β-hydroxybutyrylated peptide (the immunogen) before immunoblotting to demonstrate signal reduction
Parallel testing with other site-specific antibodies: Compare detection patterns with antibodies against other modifications at the same residue (e.g., H2A K36 acetylation)
Metabolic labeling: Treat cells with isotopically labeled β-hydroxybutyrate (e.g., 13C-labeled) followed by mass spectrometry to confirm the modification site corresponds to antibody reactivity
CRISPR-Cas9 mutagenesis: Generate K36R mutants of HIST1H2AG to create a negative control lacking the modifiable residue
Dose-dependent induction: Verify that antibody signal increases proportionally with β-hydroxybutyrate treatment concentration
This multi-faceted validation approach ensures confidence in antibody specificity and experimental results interpretation.
Optimizing ChIP-seq for β-hydroxybutyryl-HIST1H2AG (K36) requires several specialized considerations:
Crosslinking optimization: Test multiple formaldehyde concentrations (0.5-2%) and incubation times (5-20 minutes) to preserve the modification while ensuring efficient DNA fragmentation
Sonication parameters: Use gentler sonication conditions compared to standard ChIP protocols to prevent epitope damage
Antibody titration: Conduct preliminary ChIP-qPCR using different antibody amounts to determine optimal concentration for specificity and sensitivity
Include spike-in controls: Use exogenous chromatin (e.g., Drosophila) as normalization controls, particularly when comparing samples with varying global modification levels
Sequential ChIP: Consider sequential ChIP with other histone mark antibodies to identify genomic regions with co-occurring modifications
Based on previous studies, histone Kbhb marks are enriched at promoters of active genes, particularly those involved in starvation-responsive metabolic pathways . Therefore, focus initial validation on known metabolic genes regulated during fasting or ketosis.
To investigate the complex interplay between β-hydroxybutyrylation and other histone modifications, implement these methodological approaches:
Mass spectrometry-based proteomics:
Use middle-down MS approaches to analyze co-occurring modifications on the same histone tail
Apply top-down proteomics to analyze intact histone proteoforms with multiple modifications
Implement crosslinking mass spectrometry to identify protein complexes associated with multiply-modified histones
Advanced microscopy techniques:
Apply proximity ligation assays to detect co-occurrence of different modifications at the single-cell level
Use super-resolution microscopy to visualize spatial relationships between different modifications
Combinatorial biochemical approaches:
Perform sequential ChIP (re-ChIP) to identify genomic regions with co-occurring modifications
Use synthetic histone peptides with defined modification patterns in protein binding assays
Genetic and chemical manipulation:
These approaches will help decipher whether β-hydroxybutyrylation works cooperatively or competitively with other modifications in regulating gene expression.
Distinguishing between different acylation types at the same lysine residue requires sophisticated analytical approaches:
Modification-specific antibodies: Use highly specific antibodies that can differentiate between acetylation and β-hydroxybutyrylation at the same residue, validating with peptide competition assays
Mass spectrometry approaches:
Employ diagnostic fragment ions in MS/MS spectra that differentiate between acyl modifications
Use chemical derivatization strategies that selectively react with specific acyl groups
Apply targeted multiple reaction monitoring (MRM) to quantify specific modified peptides
Metabolic manipulation experiments:
Compare cells treated with β-hydroxybutyrate versus acetate to promote specific modifications
Use isotopically labeled acyl-CoA precursors to trace the incorporation of specific modifications
Enzyme selectivity assays:
Test the substrate specificity of writers (acetyltransferases vs. β-hydroxybutyryltransferases)
Examine deacylase specificity using in vitro enzymatic assays with synthetic peptides
Compare the activities of HDAC1-3 and SIRT1-3, which have been identified as enzymes capable of removing β-hydroxybutyryl groups
This systematic approach helps delineate the distinct functional consequences of different acylation types at the same residue.
Metabolic state profoundly influences histone β-hydroxybutyrylation patterns, which has significant implications for experimental design:
Fasting-induced changes:
Diabetic ketoacidosis effects:
Experimental design considerations:
Control for feeding/fasting status and time of day when collecting samples
Monitor blood β-hydroxybutyrate levels concurrently with histone modification analysis
Consider the tissue-specific responses to metabolic changes (liver shows pronounced effects)
Include both short-term and long-term metabolic interventions to distinguish acute versus chronic effects
Functional implications:
Understanding these metabolic influences is essential for designing physiologically relevant experiments and correctly interpreting results.
Current knowledge about enzymes regulating β-hydroxybutyrylation dynamics includes:
"Writers" (adding the modification):
Unlike histone acetyltransferases (HATs), specific enzymes dedicated to β-hydroxybutyrylation have not been definitively identified
Some p300/CBP enzymes may catalyze β-hydroxybutyrylation non-specifically, but this requires further validation
Non-enzymatic β-hydroxybutyrylation may occur under conditions of elevated β-hydroxybutyryl-CoA
"Erasers" (removing the modification):
HDAC1-3 and SIRT1-3 have demonstrated significant de-β-hydroxybutyrylation activity against core histones in vitro
Interestingly, only HDAC1 and HDAC2 appear to function as histone Kbhb deacetylases in cellular contexts
Deacylases may show preferences for specific chiral forms of β-hydroxybutyrylation, suggesting stereochemical regulation
"Readers" (proteins recognizing the modification):
Specific reader proteins for β-hydroxybutyrylation are still being characterized
Bromodomain-containing proteins that typically bind acetylated lysines may have different affinities for β-hydroxybutyrylated residues
Regulatory considerations:
This evolving understanding provides targets for experimental manipulation to study β-hydroxybutyrylation dynamics.
For rigorous quantitative analysis of Western blot data using β-hydroxybutyryl-HIST1H2AG (K36) Antibody, implement this methodological framework:
Technical optimization:
Use a dynamic range of protein loading (5-30 μg of histone extracts) to ensure signal linearity
Include a standard curve of recombinant β-hydroxybutyrylated peptides for absolute quantification
Process all experimental conditions on the same blot to minimize inter-blot variability
Normalization strategies:
Normalize to total histone H2A (using pan-H2A antibodies) rather than housekeeping proteins
Consider dual normalization to both total histone and loading controls
For samples with potentially altered histone levels, use spike-in controls of exogenous proteins
Quantification approaches:
Use digital imaging systems with wide dynamic range rather than film
Apply background subtraction methods consistently across all samples
Quantify integrated density values rather than peak intensity
Average technical replicates (minimum of three) before statistical analysis
Statistical analysis:
Apply appropriate statistical tests based on data distribution (parametric or non-parametric)
Consider hierarchical analysis for nested experimental designs
Report effect sizes and confidence intervals in addition to p-values
Validation considerations:
This comprehensive approach ensures reliable quantification and interpretation of β-hydroxybutyrylation dynamics.
When interpreting ChIP-seq data for histone β-hydroxybutyrylation, researchers should be aware of these common pitfalls and their solutions:
Previous studies have shown that histone Kbhb is enriched in active gene promoters and associated with starvation-responsive metabolic pathways . When analyzing your own data, consider whether your findings align with or differ from these established patterns.
When working with β-hydroxybutyryl-HIST1H2AG (K36) Antibody, researchers may encounter these common technical issues and solutions:
Weak or absent signal:
Potential causes: Insufficient modification levels, antibody degradation, epitope masking
Solutions:
High background or non-specific binding:
Potential causes: Insufficient blocking, cross-reactivity with other acylations
Solutions:
Increase blocking time or concentration (5% BSA often works better than milk for PTM antibodies)
Include additional washing steps with higher stringency buffers
Pre-absorb antibody with unmodified histone peptides
Inconsistent results between experiments:
Potential causes: Variations in metabolic state of cells, lot-to-lot antibody variability
Solutions:
Standardize culture conditions and harvest times
Test each antibody lot against a standard sample
Include positive controls in each experiment (e.g., β-hydroxybutyrate-treated cells)
Discrepancies between antibody-based detection and mass spectrometry:
Implementing these troubleshooting approaches will improve the reliability and reproducibility of your β-hydroxybutyryl-HIST1H2AG (K36) Antibody experiments.
Optimizing immunofluorescence protocols for β-hydroxybutyryl-HIST1H2AG (K36) detection in tissue sections requires specific methodological considerations:
Fixation optimization:
Test various fixatives beyond standard PFA (e.g., methanol, ethanol, or dual fixation protocols)
Limit fixation time to preserve epitope accessibility (typically 10-15 minutes for PFA)
Consider performing antigen retrieval using citrate buffer (pH 6.0) or Tris-EDTA (pH 9.0)
Permeabilization considerations:
Use gentle permeabilization methods (0.1-0.2% Triton X-100) to maintain nuclear architecture
Extend permeabilization time (15-30 minutes) to ensure antibody access to nuclear antigens
Consider alternative permeabilization agents like saponin for sensitive tissues
Blocking and antibody incubation:
Signal amplification:
Consider tyramide signal amplification for low abundance modifications
Use high-sensitivity detection systems (e.g., quantum dots or Alexa Fluor 647)
Optimize exposure settings to capture the full dynamic range without saturation
Controls and validation:
These optimizations will help achieve specific and sensitive detection of β-hydroxybutyrylation in tissue sections.
Several promising research areas for exploring the functional significance of histone β-hydroxybutyrylation in disease models include:
Metabolic disorders:
Investigate how altered β-hydroxybutyrylation patterns contribute to transcriptional dysregulation in obesity and diabetes
Explore whether β-hydroxybutyrylation changes precede or follow insulin resistance development
Analyze tissue-specific differences in modification patterns across metabolic disease progression
Neurodegenerative diseases:
Examine β-hydroxybutyrylation in ketogenic diet treatments for epilepsy and neurodegenerative conditions
Investigate whether β-hydroxybutyrylation mediates neuroprotective effects of ketone bodies
Study how age-related changes in histone β-hydroxybutyrylation affect neuronal gene expression
Cancer metabolism:
Explore how metabolic reprogramming in cancer cells affects histone β-hydroxybutyrylation
Investigate whether targeting β-hydroxybutyrylation pathways can sensitize cancer cells to therapy
Analyze tumor microenvironment effects on cancer cell β-hydroxybutyrylation patterns
Inflammatory conditions:
Study how β-hydroxybutyrate's anti-inflammatory effects may be mediated through histone β-hydroxybutyrylation
Examine modification patterns in macrophages during polarization and inflammatory responses
Investigate potential targeting of β-hydroxybutyrylation pathways for inflammatory disease treatment
Developmental programming:
These research directions could reveal how this metabolically-responsive histone modification contributes to disease pathogenesis and identify potential therapeutic targets.
Integrating computational approaches and systems biology into histone β-hydroxybutyrylation research offers powerful methodological frameworks:
Multi-omics data integration:
Develop computational pipelines that integrate ChIP-seq, RNA-seq, and metabolomics data
Apply machine learning algorithms to identify patterns linking metabolic state, histone modifications, and gene expression
Implement network analysis to map the relationships between modified histones and transcriptional programs
Predictive modeling:
Develop algorithms to predict β-hydroxybutyrylation sites based on sequence context and chromatin features
Create mathematical models of the dynamics between β-hydroxybutyrate metabolism and histone modification
Simulate the effects of metabolic perturbations on global modification patterns
Comparative epigenomics:
Apply phylogenetic analysis to understand the evolutionary conservation of β-hydroxybutyrylation sites
Compare modification patterns across species under similar metabolic states
Identify species-specific regulatory mechanisms through comparative genomics
Structure-function relationships:
Use molecular dynamics simulations to predict how β-hydroxybutyrylation affects chromatin fiber structure
Model reader protein interactions with modified histones through docking simulations
Predict the effects of β-hydroxybutyrylation on nucleosome stability and dynamics
Clinical data mining:
These computational approaches will accelerate discovery and provide mechanistic insights into this emerging epigenetic regulatory mechanism.
A comparative analysis of antibodies targeting different β-hydroxybutyrylation sites reveals important distinctions:
Feature | β-hydroxybutyryl-HIST1H2AG (K36) | 2-hydroxyisobutyryl-HIST1H2AG (K95) | 2-hydroxyisobutyryl-HIST1H2AG (K36) |
---|---|---|---|
Host Species | Rabbit | Rabbit | Rabbit |
Clonality | Polyclonal | Polyclonal | Polyclonal |
Validated Applications | ELISA, WB | ELISA, IF | ELISA, ICC |
Recommended Dilutions | Varies by application | IF: 1:50-1:200 | Application-specific |
Buffer Composition | Not specified | 50% Glycerol, 0.01M PBS, pH 7.4, 0.03% Proclin 300 | Not specified |
Storage Conditions | -20°C to -80°C | -20°C to -80°C, avoid repeated freeze-thaw | -20°C to -80°C |
Chromatin Context | Active gene promoters | Less characterized | Less characterized |
Metabolic Response | Strongly induced by fasting/ketosis | Requires further characterization | Requires further characterization |
Key considerations when selecting between these antibodies:
The biological question (specific modification site relevance)
Required applications (WB vs. IF/ICC capabilities)
Chromatin context of interest (promoters vs. other regions)
Understanding these differences enables researchers to select the most appropriate antibody for their specific experimental needs.
When working with β-hydroxybutyryl versus 2-hydroxyisobutyryl antibodies, researchers should consider these methodological differences:
Structural recognition differences:
β-hydroxybutyryl modifications contain a straight-chain structure with a β-hydroxyl group
2-hydroxyisobutyryl modifications contain a branched structure with a 2-hydroxyl group
These structural differences affect antibody specificity and may require different blocking strategies
Application-specific considerations:
Metabolic induction protocols:
β-hydroxybutyrylation is specifically induced by β-hydroxybutyrate treatment
2-hydroxyisobutyrylation may respond differently to metabolic stimuli
Researchers should validate the appropriate metabolite treatment for each modification
Cross-reactivity concerns:
Due to structural similarities, validation of specificity between these closely related modifications is essential
Include appropriate competition controls with modified and unmodified peptides
Enzymatic regulation:
Understanding these methodological differences will help researchers design experiments that accurately distinguish between these related but distinct histone modifications.
Recent significant advances in histone β-hydroxybutyrylation research include:
Comprehensive identification of modification sites:
Metabolic regulation mechanisms:
Enzymatic regulation:
Functional genomics insights:
Key questions that remain unanswered:
Dedicated "writers" - Are there specific enzymes that preferentially catalyze β-hydroxybutyrylation, or is it primarily non-enzymatic?
Selective "readers" - Which proteins specifically recognize β-hydroxybutyrylated histones, and how does this recognition differ from other acylations?
Tissue specificity - How do β-hydroxybutyrylation patterns differ across tissues, and what are the functional consequences?
Disease relevance - How are β-hydroxybutyrylation patterns altered in specific diseases, and could targeting these pathways offer therapeutic benefits?
Evolutionary conservation - How conserved are the functions of β-hydroxybutyrylation across species, and what does this reveal about its fundamental importance?
Addressing these questions represents a significant opportunity for researchers to advance our understanding of this metabolically-responsive epigenetic mechanism.
Emerging technologies poised to transform histone β-hydroxybutyrylation research include:
Advanced mass spectrometry approaches:
Ion mobility MS for improved separation of modified peptides
Targeted SWATH-MS for comprehensive quantification across multiple samples
Single-cell proteomics to reveal cell-to-cell variation in modification patterns
Genome editing technologies:
Base editing to precisely modify lysine residues without disrupting histone genes
CRISPR activation/interference systems to modulate enzymes involved in β-hydroxybutyrylation
Prime editing for introducing specific mutations in histone genes
Live-cell imaging innovations:
FRET-based sensors for real-time monitoring of β-hydroxybutyrylation dynamics
Engineered antibody fragments for live-cell modification tracking
Super-resolution microscopy combined with specific probes for spatial organization
Synthetic biology approaches:
Engineered histone reader domains with specificity for β-hydroxybutyrylation
Orthogonal synthetic systems to control modification levels independently of metabolism
Designer nucleosomes with defined modification patterns for biochemical studies
Microfluidic and organ-on-chip technologies: