β-hydroxybutyryl-HIST1H2AG (K36) Antibody

What is β-hydroxybutyrylation and why is it significant?

β-hydroxybutyrylation (Kbhb) is a post-translational modification of lysine residues in histones that was identified and verified as a new type of histone mark. This modification is particularly significant because it represents a direct link between metabolism and gene expression. β-hydroxybutyrylation marks are dramatically induced in response to elevated β-hydroxybutyrate levels, which occur during metabolic states such as prolonged fasting and diabetic ketoacidosis. With 44 identified histone Kbhb sites (comparable to the number of known histone acetylation sites), this modification represents an important epigenetic regulatory mark that offers new avenues to study chromatin regulation in the context of human pathophysiological states, including diabetes, epilepsy, and neoplasia .

What is the biochemical basis for β-hydroxybutyrylation?

The biochemical mechanism of β-hydroxybutyrylation involves the modification of lysine residues by β-hydroxybutyryl-CoA, which serves as the cofactor for this reaction. This is analogous to how acetyl-CoA serves as a cofactor for histone acetylation. Research has demonstrated that β-hydroxybutyryl-CoA can be generated from cellular β-hydroxybutyrate, possibly through the action of short-chain-Coenzyme A synthetase. This process has been verified through metabolic labeling experiments using isotopically labeled sodium β-hydroxybutyrate, which confirmed that sodium β-hydroxybutyrate can be converted into β-hydroxybutyryl-CoA in cells, which then serves as the donor for the lysine modification .

What is the β-hydroxybutyryl-HIST1H2AG (K36) antibody and what are its basic characteristics?

The β-hydroxybutyryl-HIST1H2AG (K36) antibody is a polyclonal antibody raised in rabbits that specifically recognizes the β-hydroxybutyrylation modification at lysine 36 of histone H2A type 1 (HIST1H2AG) in humans. This primary antibody has IgG isotype and is typically supplied in an unconjugated form. The antibody was generated using a peptide sequence around the site of β-hydroxybutyryl-Lys (36) derived from Human Histone H2A type 1 as the immunogen. It has been validated for applications including Enzyme-Linked Immunosorbent Assay (ELISA) and Western Blot (WB) .

How can I validate the specificity of the β-hydroxybutyryl-HIST1H2AG (K36) antibody?

To validate the specificity of the β-hydroxybutyryl-HIST1H2AG (K36) antibody, researchers should consider the following methodological approach:

• Dot blot assay: Perform a dot blot assay using synthetic peptides with various modifications (β-hydroxybutyrylation, acetylation, 2-hydroxyisobutyrylation) at the K36 position of HIST1H2AG. Compare signal intensity with the target antibody.

• Competition experiments: Conduct competition experiments where the antibody is pre-incubated with excess synthetic β-hydroxybutyrylated peptides before immunoblotting to confirm specificity.

• Western blot analysis with controls: Use histones from cells treated with and without β-hydroxybutyrate, comparing with other known histone modification antibodies to ensure specific recognition.

• Mass spectrometry validation: Confirm the presence of the β-hydroxybutyrylation mark at K36 in immunoprecipitated histones using high-resolution mass spectrometry.

These approaches, similar to those used for pan anti-Kbhb antibody validation, will ensure the antibody specifically recognizes the β-hydroxybutyrylation mark at K36 of HIST1H2AG .

What are the optimal conditions for using β-hydroxybutyryl-HIST1H2AG (K36) antibody in Western blotting experiments?

For optimal Western blotting results with the β-hydroxybutyryl-HIST1H2AG (K36) antibody, follow these methodological guidelines:

• Sample preparation: Extract histones using acid extraction methods to ensure enrichment of histone proteins. For cellular experiments examining β-hydroxybutyrylation induction, treat cells with sodium β-hydroxybutyrate at concentrations ranging from 1-10 mM for 12-24 hours before extraction.

• Gel electrophoresis: Use 15-18% SDS-PAGE gels which are optimal for resolving histone proteins.

• Transfer conditions: Employ PVDF membranes with low pore size (0.2 μm) and implement wet transfer at low voltage (30V) overnight at 4°C for efficient transfer of small histone proteins.

• Blocking: Block membranes with 5% non-fat dry milk or BSA in TBST for 1 hour at room temperature.

• Antibody dilution: The optimal dilution should be determined empirically, but typically starts at 1:1000 in blocking buffer.

• Controls: Include both positive controls (histones from cells treated with β-hydroxybutyrate) and negative controls (histones from cells where β-hydroxybutyrylation is minimized).

• Detection: Use appropriate HRP-conjugated secondary antibodies and enhanced chemiluminescence detection systems .

How can I design experiments to study the dynamic changes of β-hydroxybutyrylation at HIST1H2AG (K36) in response to metabolic changes?

To study dynamic changes in β-hydroxybutyrylation at HIST1H2AG (K36) in response to metabolic changes, consider this experimental design:

• Metabolic manipulation models:

  • In vitro: Treat cells with varying concentrations of sodium β-hydroxybutyrate (1-10 mM) for different time periods

  • Ex vivo: Use primary cells or tissues from models of fasting (12-48 hours) or diabetic ketoacidosis

  • In vivo: Employ animal models subjected to fasting, ketogenic diet, or streptozotocin-induced diabetes

• Assessment approaches:

  • Western blotting: Quantify β-hydroxybutyrylation levels using the β-hydroxybutyryl-HIST1H2AG (K36) antibody

  • Chromatin immunoprecipitation (ChIP): Map the genomic distribution of H2AK36bhb using the antibody

  • Immunofluorescence: Visualize nuclear localization and intensity of the modification

• Time-course analysis: Monitor changes at multiple time points to establish the temporal dynamics of the modification

• Correlation analysis: Measure serum β-hydroxybutyrate levels concurrently and correlate with H2AK36bhb levels

• Molecular intervention: Use HDAC inhibitors or β-hydroxybutyrate metabolism inhibitors to modulate the dynamics

This comprehensive approach will allow for detailed characterization of how β-hydroxybutyrylation at HIST1H2AG (K36) responds to different metabolic states .

How can I use β-hydroxybutyryl-HIST1H2AG (K36) antibody for ChIP-seq experiments to map genome-wide distribution?

For conducting ChIP-seq experiments with β-hydroxybutyryl-HIST1H2AG (K36) antibody, follow this methodological framework:

• Crosslinking and chromatin preparation:

  • Crosslink cells with 1% formaldehyde for 10 minutes at room temperature

  • Quench with 125 mM glycine for 5 minutes

  • Isolate nuclei and sonicate chromatin to obtain fragments of 200-500 bp

  • Verify fragment size by agarose gel electrophoresis

• Immunoprecipitation optimization:

  • Determine optimal antibody concentration through titration experiments (typically 2-5 μg per ChIP reaction)

  • Include appropriate controls: IgG negative control, input chromatin, and a positive control antibody against a well-characterized histone mark

• ChIP-seq specific considerations:

  • Ensure sufficient depth of sequencing (minimum 20 million unique reads)

  • Include spike-in controls for normalization across different metabolic conditions

  • Perform replicate experiments (minimum of 3 biological replicates)

• Bioinformatic analysis:

  • Map reads to the genome using appropriate alignment tools

  • Call peaks using algorithms suitable for histone modifications (e.g., MACS2)

  • Perform differential binding analysis between experimental conditions

  • Correlate binding patterns with gene expression data and other histone marks

• Validation:

  • Confirm selected peak regions by ChIP-qPCR

  • Perform sequential ChIP (re-ChIP) to examine co-occurrence with other histone marks

Based on previous studies of histone β-hydroxybutyrylation, this approach should reveal that H2AK36bhb is likely enriched in active gene promoters and associated with starvation-responsive metabolic pathways .

What is the relationship between β-hydroxybutyrylation and other histone modifications, and how can this be studied?

The relationship between β-hydroxybutyrylation and other histone modifications requires sophisticated experimental approaches:

• Sequential ChIP (re-ChIP) analysis:

  • Perform primary ChIP with β-hydroxybutyryl-HIST1H2AG (K36) antibody

  • Elute the immunoprecipitated chromatin

  • Perform secondary ChIP with antibodies against other modifications

  • Analyze regions of co-occurrence or mutual exclusivity

• Mass spectrometry-based combinatorial analysis:

  • Implement middle-down or top-down proteomics approaches

  • Analyze histone peptides containing K36 and nearby modification sites

  • Quantify the relative abundance of different modification combinations

• Multi-omics integration:

  • Correlate ChIP-seq data for β-hydroxybutyrylation with datasets for other histone marks

  • Employ machine learning algorithms to identify patterns and relationships

• Functional perturbation experiments:

  • Manipulate writer or eraser enzymes for specific modifications

  • Assess the impact on β-hydroxybutyrylation levels

  • Use histone mutants to investigate modification crosstalk

• Temporal dynamics analysis:

  • Monitor changes in multiple modifications during metabolic transitions

  • Determine the sequential order of appearance/disappearance

Previous research suggests that while some modifications show coordinated regulation with β-hydroxybutyrylation (particularly in response to metabolic changes), others may be independently regulated. For example, during prolonged fasting, histone Kbhb levels were significantly elevated while histone acetylation showed little to no change, suggesting distinct regulatory mechanisms for these modifications .

How can I determine the enzymes responsible for writing and erasing β-hydroxybutyrylation at HIST1H2AG (K36)?

To identify enzymes involved in β-hydroxybutyrylation dynamics at HIST1H2AG (K36), implement this comprehensive strategy:

• Candidate enzyme screening:

  • Conduct in vitro enzymatic assays with recombinant histone acetyltransferases (HATs) and histone deacetylases (HDACs) to test their activity toward β-hydroxybutyrylated histones

  • Focus on enzymes known to target K36 of H2A for other modifications

  • Screen sirtuins (especially SIRT1-7) which have been implicated in removing various acylations

• Genetic manipulation approaches:

  • Perform siRNA/shRNA knockdown or CRISPR-Cas9 knockout of candidate enzymes

  • Overexpress wild-type and catalytically inactive mutants of candidate enzymes

  • Quantify changes in H2AK36bhb levels by Western blotting and immunofluorescence

• Protein-protein interaction studies:

  • Conduct co-immunoprecipitation experiments to identify proteins interacting with β-hydroxybutyrylated H2A

  • Use proximity ligation assays to confirm interactions in situ

  • Employ BioID or APEX2 proximity labeling to identify proteins in close proximity to H2AK36bhb

• Enzyme kinetics and substrate specificity:

  • For identified candidates, perform detailed enzyme kinetics with synthetic peptide substrates

  • Compare activity on β-hydroxybutyrylated versus other modified lysine residues

• In vivo validation:

  • Generate mouse models with tissue-specific knockout of identified enzymes

  • Analyze H2AK36bhb levels during metabolic challenges like fasting

This systematic approach should reveal the enzymatic machinery responsible for the dynamic regulation of β-hydroxybutyrylation at HIST1H2AG (K36), providing critical insights into the metabolic regulation of this epigenetic mark .

What are the common technical challenges when using β-hydroxybutyryl-HIST1H2AG (K36) antibody and how can they be addressed?

Researchers commonly encounter several technical challenges when working with the β-hydroxybutyryl-HIST1H2AG (K36) antibody. Here are methodological solutions to address these issues:

• High background signal in Western blots:

  • Increase blocking time or concentration (try 5% BSA instead of milk)

  • Reduce primary antibody concentration (perform titration series)

  • Increase washing steps (5 washes of 5-10 minutes each)

  • Add 0.1-0.5% Triton X-100 to washing buffer

  • Pre-adsorb antibody with unmodified histone proteins

• Weak or no signal detection:

  • Ensure target modification is present (treat cells with β-hydroxybutyrate)

  • Optimize histone extraction protocol to preserve modifications

  • Decrease antibody dilution

  • Increase exposure time for detection

  • Use signal enhancement systems

• Cross-reactivity with other modifications:

  • Perform peptide competition assays with β-hydroxybutyrylated and other modified peptides

  • Compare signal with other specific histone modification antibodies

  • Validate by mass spectrometry

• Inconsistent ChIP-seq results:

  • Optimize chromatin fragmentation conditions

  • Increase antibody amount (up to 5 μg per reaction)

  • Extend incubation time (overnight at 4°C)

  • Include spike-in controls for normalization

• Sample degradation issues:

  • Add histone deacetylase inhibitors (e.g., sodium butyrate) to all buffers

  • Work at 4°C throughout the procedure

  • Add protease inhibitor cocktails

  • Minimize freeze-thaw cycles of antibody

These optimization strategies should improve the reliability and reproducibility of experiments using the β-hydroxybutyryl-HIST1H2AG (K36) antibody .

How can I distinguish between β-hydroxybutyrylation and structurally similar modifications?

Distinguishing between β-hydroxybutyrylation and structurally similar modifications like 2-hydroxyisobutyrylation requires a multi-faceted analytical approach:

• High-resolution mass spectrometry:

  • Employ high-resolution LC-MS/MS to differentiate between modification isomers

  • Compare retention times with synthetic peptides containing different modifications

  • Analyze fragmentation patterns which will differ between isomeric modifications

  • Use heavy isotope-labeled standards for accurate identification

• Antibody specificity validation:

  • Perform dot blot assays with synthetic peptides bearing different modifications

  • Test antibody cross-reactivity with peptides containing:

    • β-hydroxybutyrylation (K-bhb)

    • 2-hydroxyisobutyrylation (K-2hib)

    • Butyrylation (K-bu)

    • Crotonylation (K-cr)

    • Other structural isomers (K-2hb, K-3hb, K-4hb)

• Metabolic labeling experiments:

  • Treat cells with isotopically labeled precursors specific to each modification

  • Track the incorporation of labels into histone PTMs

• Modification-specific enzymatic removal:

  • Identify and utilize enzymes with specificity for particular acylations

  • Measure resistance/susceptibility to specific enzyme treatments

As demonstrated in previous research, synthetic peptides containing different modification isomers (K-bhb, K-2hib, K-2hb, K-4hb) showed distinct HPLC retention times and MS/MS fragmentation patterns, providing reliable methods for distinguishing these modifications .

How can I quantify changes in β-hydroxybutyrylation levels at HIST1H2AG (K36) across different experimental conditions?

For precise quantification of β-hydroxybutyrylation changes at HIST1H2AG (K36), implement these methodological approaches:

• Quantitative Western blotting:

  • Use dual-color fluorescent Western blotting with β-hydroxybutyryl-HIST1H2AG (K36) antibody and a total H2A antibody

  • Normalize β-hydroxybutyrylation signal to total H2A signal

  • Include a standard curve using recombinant modified histones

  • Employ image analysis software for densitometry

• Mass spectrometry-based quantification:

  • Implement label-free quantification (LFQ) of tryptic peptides

  • Use stable isotope labeling (SILAC) for comparing different conditions

  • Apply multiple reaction monitoring (MRM) for targeted quantification

  • Calculate the stoichiometry of modification by comparing modified and unmodified peptides

• ChIP-qPCR for locus-specific quantification:

  • Design primers for regions of interest identified from ChIP-seq

  • Normalize to input DNA and control regions

  • Calculate fold enrichment over IgG control

• ELISA-based approaches:

  • Develop sandwich ELISA using anti-H2A capture antibody and β-hydroxybutyryl-HIST1H2AG (K36) detection antibody

  • Generate standard curves with synthetic modified peptides

• Data presentation and statistical analysis:

  • Present data as fold change relative to control conditions

  • Perform appropriate statistical tests (t-test for two conditions, ANOVA for multiple conditions)

  • Ensure biological replicates (n ≥ 3) for robust analysis

This multimodal approach ensures accurate quantification of β-hydroxybutyrylation levels across experimental conditions, enabling reliable interpretation of the biological significance of observed changes .

What are the functional implications of β-hydroxybutyrylation at HIST1H2AG (K36) in gene regulation?

The functional implications of β-hydroxybutyrylation at HIST1H2AG (K36) in gene regulation can be explored through these methodological approaches:

• Integrative genomics analysis:

  • Correlate H2AK36bhb ChIP-seq profiles with transcriptome data

  • Identify gene ontology categories and pathways enriched among H2AK36bhb-associated genes

  • Compare genomic distribution with known regulatory elements (promoters, enhancers)

• Mechanistic studies of transcriptional regulation:

  • Investigate recruitment of transcription factors and cofactors to H2AK36bhb-enriched regions

  • Examine chromatin accessibility changes (ATAC-seq) in response to β-hydroxybutyrylation changes

  • Assess the impact on RNA polymerase II occupancy and elongation

• Functional genomics approaches:

  • Implement CRISPR-Cas9 to mutate K36 to non-modifiable residues in H2A

  • Measure consequent changes in target gene expression

  • Create synthetic modified nucleosomes for in vitro transcription assays

Based on research on histone β-hydroxybutyrylation more broadly, this modification is likely enriched in active gene promoters and associated with starvation-responsive metabolic pathways. The specific role of H2AK36bhb may involve creating binding surfaces for specific reader proteins that facilitate transcriptional activation of metabolically regulated genes .

How is β-hydroxybutyrylation at HIST1H2AG (K36) involved in metabolic diseases and potential therapeutic approaches?

The involvement of β-hydroxybutyrylation at HIST1H2AG (K36) in metabolic diseases and its therapeutic potential can be investigated through:

• Clinical sample analysis:

  • Compare H2AK36bhb levels in tissues from healthy individuals versus patients with metabolic disorders (diabetes, obesity)

  • Correlate modification levels with clinical parameters (glucose levels, ketone bodies)

  • Perform longitudinal studies during disease progression

• Disease model characterization:

  • Analyze H2AK36bhb in animal models of diabetes, obesity, and ketogenic states

  • Perform ChIP-seq to identify differentially marked genes in disease states

  • Correlate with transcriptomic and metabolomic changes

• Intervention studies:

  • Test the effects of drugs that modulate β-hydroxybutyrate metabolism

  • Investigate ketogenic diets or fasting regimens on H2AK36bhb patterns

  • Target identified writer/eraser enzymes with small molecule inhibitors

• Therapeutic development framework:

  • Screen compound libraries for molecules that specifically modulate H2AK36bhb

  • Develop targeted degradation approaches for specific reader proteins

  • Explore β-hydroxybutyrate analogs as potential epigenetic modifiers

Given that histone β-hydroxybutyrylation connects metabolism to gene expression and is dramatically induced during fasting and diabetic ketoacidosis, therapeutic approaches targeting this modification could potentially address metabolic disorders by reprogramming gene expression patterns in a metabolism-sensitive manner .

How does β-hydroxybutyrylation at HIST1H2AG (K36) compare to this modification at other histone residues?

A comparative analysis of β-hydroxybutyrylation at HIST1H2AG (K36) versus other histone residues requires:

• Comprehensive profiling approach:

  • Perform mass spectrometry-based proteomics to identify and quantify β-hydroxybutyrylation at different histone residues

  • Develop residue-specific antibodies for various Kbhb sites

  • Compare relative abundance and dynamics across different metabolic conditions

• ChIP-seq comparative analysis:

  • Conduct parallel ChIP-seq experiments for different Kbhb-modified histones

  • Compare genomic distribution patterns

  • Identify unique and overlapping target genes

• Functional hierarchy determination:

  • Perform sequential genetic modifications of different Kbhb sites

  • Assess relative contributions to transcriptional outcomes

  • Identify possible synergistic or antagonistic relationships

• Structural biology investigations:

  • Compare the structural impacts of β-hydroxybutyrylation at different residues

  • Identify residue-specific reader proteins using peptide pull-down assays

  • Determine crystal structures of reader domains bound to different Kbhb-modified histone peptides

Previous research has identified 44 histone Kbhb sites, including modifications on H3K4, H3K9, H3K56, H4K8, and H4K12. These sites are likely to have distinct functions based on their locations within the nucleosome structure and their proximity to other regulatory elements. For example, H3K9bhb might directly affect transcriptional activation, while H2AK36bhb could influence nucleosome stability or higher-order chromatin structure .