β-hydroxybutyryl-HIST1H2BC (K120) Antibody

What is β-hydroxybutyryl-HIST1H2BC (K120) Antibody and what modification does it detect?

The β-hydroxybutyryl-HIST1H2BC (K120) Antibody is a polyclonal antibody produced in rabbits that specifically recognizes the β-hydroxybutyryl modification at lysine 120 of the histone protein HIST1H2BC. This antibody detects a relatively newly discovered histone post-translational modification involved in gene regulation and chromatin structure. The antibody specifically binds to the peptide sequence surrounding the β-hydroxybutyrylated lysine 120 residue derived from Human Histone H2B type 1-C/E/F/G/I . The modification is part of a growing family of acylation marks on histones that play critical roles in epigenetic regulation.

How does β-hydroxybutyrylation differ from other histone modifications like acetylation?

β-hydroxybutyrylation (Kbhb) is distinguishable from acetylation and other histone modifications in several key ways:

• Metabolic origin: β-hydroxybutyrylation is directly linked to cellular metabolism, specifically β-hydroxybutyrate levels, which increase during fasting, ketogenic diets, and diabetic ketoacidosis .

• Regulatory dynamics: Unlike acetylation, which shows minimal changes during metabolic shifts, histone Kbhb levels dramatically increase in response to elevated β-hydroxybutyrate concentrations. Research has demonstrated that while histone acetylation shows little change during fasting or in diabetic models, Kbhb increases substantially (up to 10-fold in some cases) .

• Structural differences: The β-hydroxybutyryl group is larger than the acetyl group and contains a hydroxyl group, potentially allowing for additional hydrogen bonding interactions with reader proteins.

• Gene regulation specificity: Evidence suggests that β-hydroxybutyrylation marks may regulate a distinct set of genes compared to acetylation, particularly those involved in metabolic adaptation during fasting or ketotic states .

Why is the K120 site of HIST1H2BC significant for research?

The K120 site on HIST1H2BC represents a critical position for several reasons:

• Evolutionary conservation: This lysine residue is highly conserved across species, suggesting functional importance.

• Structural significance: K120 is located in a region of the histone that influences nucleosome stability and chromatin architecture.

• Regulatory potential: Modification at K120 may affect the interaction between histones and DNA, potentially influencing gene accessibility and transcription.

• Disease relevance: Aberrant modification at this site may be involved in disease processes, particularly those with metabolic components such as diabetes and cancer .

What are the validated applications for β-hydroxybutyryl-HIST1H2BC (K120) Antibody?

The β-hydroxybutyryl-HIST1H2BC (K120) Antibody has been validated for:

• Western Blotting (WB): Recommended dilution range of 1:100-1:1000, allowing detection of the modification in whole cell lysates from various human cell lines (A549, K562, HepG2) .

• Enzyme-Linked Immunosorbent Assay (ELISA): Recommended dilution range of 1:2000-1:10000 for detection of the specific modification .

• Potential application in Chromatin Immunoprecipitation (ChIP): While not explicitly validated for the K120 antibody, similar antibodies targeting β-hydroxybutyrylation at other sites have been successfully used in ChIP experiments to map genomic locations of the modification .

How should I optimize Western blot protocols for detection of β-hydroxybutyryl-HIST1H2BC (K120)?

For optimal Western blot detection of β-hydroxybutyryl-HIST1H2BC (K120):

• Sample preparation:

  • Extract histones using an acid extraction method to enrich for histone proteins

  • Use fresh samples or store extracted histones at -80°C to preserve modifications

  • Include protease inhibitors and deacetylase inhibitors (such as sodium butyrate at 30mM) in extraction buffers

• Gel electrophoresis and transfer:

  • Use 15-18% SDS-PAGE gels for optimal histone separation

  • Transfer to PVDF membranes (rather than nitrocellulose) for better retention of histones

  • Use low methanol transfer buffers to improve transfer efficiency of basic proteins

• Blocking and antibody incubation:

  • Block with 5% BSA rather than milk (milk contains histones that may cause background)

  • Use the antibody at 1:500 dilution initially, then optimize based on signal strength

  • Incubate overnight at 4°C for maximal sensitivity

• Positive controls:

  • Include lysates from cells treated with 10-30 mM sodium β-hydroxybutyrate for 4-6 hours as a positive control

  • Consider using lysates from fasted animal tissues (particularly liver) which naturally have elevated levels of this modification

How can I induce and detect changes in β-hydroxybutyrylation levels in experimental systems?

Several approaches can be used to modulate and detect β-hydroxybutyrylation levels:

• Metabolic induction methods:

  • Treat cells with sodium β-hydroxybutyrate (5-30 mM) for 4-24 hours to induce dose-dependent increases in histone Kbhb

  • Use isotopically labeled β-hydroxybutyrate (13C2) to confirm the direct incorporation into histone modifications

  • Employ fasting protocols in animal models (16-24 hours) to naturally elevate β-hydroxybutyrate levels and histone Kbhb

• Disease models:

  • The streptozotocin (STZ)-induced Type 1 diabetes mellitus mouse model shows dramatically elevated histone Kbhb levels (10-fold elevation in blood β-hydroxybutyrate correlates with significant increases in histone Kbhb)

• Detection methods:

  • Western blotting with site-specific antibodies provides a targeted approach

  • Mass spectrometry (HPLC/MS/MS) allows comprehensive identification of β-hydroxybutyrylation sites

  • ChIP-seq can map genome-wide distribution of the modification

Table 1: Comparison of experimental conditions and their effects on histone β-hydroxybutyrylation levels

How does β-hydroxybutyrylation at K120 compare with other β-hydroxybutyrylation sites on histones?

Multiple β-hydroxybutyrylation sites have been identified across various histones, with different functional implications:

• Distribution pattern:

  • H2B histone proteins have multiple β-hydroxybutyrylation sites, including K12, K20, and K120, suggesting a complex regulatory pattern

  • Comprehensive mass spectrometry studies have identified at least 44 histone Kbhb sites across all core histones

• Comparative analysis:

  • Different sites show varied responses to metabolic changes, with certain sites (like H2A K5) showing up to 33-fold increases during fasting while others show more modest changes

  • The K120 site on HIST1H2BC appears to be particularly responsive to metabolic fluctuations based on antibody detection in Western blots

• Site-specific regulation:

  • The specific location of β-hydroxybutyrylation within the histone tail or core domain influences its impact on chromatin structure

  • K120 is positioned at a critical interface region that may affect nucleosome stability differently than modifications at other sites

Table 2: Selected histone β-hydroxybutyrylation sites with their response to fasting conditions

What is known about the enzymatic regulation of β-hydroxybutyrylation at K120?

The enzymatic regulation of β-hydroxybutyrylation is still being elucidated, but current understanding suggests:

• Writers (enzymes that add the modification):

  • The specific enzymes responsible for adding β-hydroxybutyryl groups to histones are not fully characterized

  • Evidence suggests that some histone acetyltransferases (HATs) may possess promiscuous activity allowing them to utilize β-hydroxybutyryl-CoA as a substrate

  • The non-enzymatic addition of β-hydroxybutyryl groups may also occur at high β-hydroxybutyrate concentrations

• Erasers (enzymes that remove the modification):

  • Certain histone deacetylases (HDACs), particularly the sirtuin family, may remove β-hydroxybutyryl groups

  • HDAC inhibition with compounds like sodium butyrate increases β-hydroxybutyrylation levels, suggesting shared enzymatic regulation with acetylation

• Readers (proteins that recognize the modification):

  • The specific reader proteins that recognize β-hydroxybutyrylation at K120 are still being identified

  • Bromodomain-containing proteins that typically bind acetylated lysines may also recognize β-hydroxybutyrylated lysines with different affinities

How might β-hydroxybutyrylation at K120 interact with or influence other histone modifications?

The interplay between β-hydroxybutyrylation and other histone modifications represents a complex regulatory network:

• Competitive modification:

  • The K120 site can potentially undergo various modifications including acetylation, methylation, and ubiquitination

  • β-hydroxybutyrylation may compete with these modifications, creating a dynamic regulatory switch depending on metabolic conditions

• Cross-talk mechanisms:

  • The presence of β-hydroxybutyrylation at K120 may influence the deposition or removal of other modifications at nearby residues

  • This creates potential for combinatorial patterns that specify unique chromatin states

• Functional consequences:

  • The specific combination of β-hydroxybutyrylation at K120 with other modifications likely regulates distinct sets of genes

  • During metabolic stress or ketogenic states, β-hydroxybutyrylation may override other modifications to activate metabolic adaptation genes

• Evolutionary implications:

  • The conservation of this modification site across species suggests an important role in fundamental chromatin processes

  • The interaction with other modifications may represent an evolutionarily conserved mechanism for metabolic adaptation

How can I verify the specificity of β-hydroxybutyryl-HIST1H2BC (K120) Antibody in my experiments?

Verifying antibody specificity is crucial for reliable results:

• Positive controls:

  • Use lysates from cells treated with sodium β-hydroxybutyrate (30mM for 4 hours)

  • Include samples from fasted animals or diabetic models known to have elevated β-hydroxybutyrylation

• Negative controls:

  • Include untreated cell lysates as baseline comparisons

  • Consider using histone demethylase or deacetylase inhibitors to create differential modification patterns

• Competition assays:

  • Pre-incubate the antibody with excess β-hydroxybutyrylated peptide corresponding to the K120 site

  • A true specific antibody will show diminished signal when pre-blocked with the target peptide

• Cross-reactivity testing:

  • Test against peptides with similar modifications (acetylation, propionylation) at the same site

  • Test against the same modification (β-hydroxybutyrylation) at different lysine residues

• Mass spectrometry validation:

  • Confirm the presence and changes in β-hydroxybutyrylation using HPLC/MS/MS analysis of immunoprecipitated histones

What are common issues when detecting β-hydroxybutyrylation and how can they be resolved?

Researchers may encounter several challenges when working with β-hydroxybutyryl-HIST1H2BC (K120) Antibody:

• Weak signal issues:

  • Problem: Low detection signal in Western blots

  • Solutions:

    • Enrich for histones using acid extraction

    • Increase antibody concentration (1:100-1:500)

    • Extend primary antibody incubation time (overnight at 4°C)

    • Use enhanced chemiluminescence detection systems

    • Include 30mM sodium butyrate in lysis buffers to prevent deacylation during extraction

• High background issues:

  • Problem: Non-specific binding creating background noise

  • Solutions:

    • Use BSA instead of milk for blocking

    • Increase washing steps and duration

    • Optimize antibody dilution (try 1:1000-1:2000)

    • Use more stringent washing buffers (increase Tween-20 concentration slightly)

• Cross-reactivity concerns:

  • Problem: Potential detection of other modifications

  • Solutions:

    • Validate with peptide competition assays

    • Compare with mass spectrometry data

    • Use multiple antibodies targeting different epitopes of the same modification

• Sample degradation:

  • Problem: Loss of modification during sample processing

  • Solutions:

    • Add deacetylase inhibitors (such as sodium butyrate) to all buffers

    • Process samples quickly and keep cold

    • Avoid freeze-thaw cycles of prepared samples

How should I interpret changes in β-hydroxybutyrylation levels across different experimental conditions?

Proper interpretation of β-hydroxybutyrylation data requires consideration of several factors:

• Metabolic context interpretation:

  • Increased β-hydroxybutyrylation generally correlates with elevated β-hydroxybutyrate levels

  • Consider measuring blood or media β-hydroxybutyrate concentrations alongside histone modifications

  • Remember that a 10-fold increase in β-hydroxybutyrate can lead to dramatic increases in histone β-hydroxybutyrylation

• Comparative analysis approach:

  • Always compare β-hydroxybutyrylation changes with other histone modifications (especially acetylation)

  • While acetylation may remain relatively unchanged, β-hydroxybutyrylation can show significant fluctuations in response to metabolic shifts

  • Different histone sites show varied sensitivity to β-hydroxybutyrylation (ranging from 10-fold to 33-fold increases)

• Functional significance assessment:

  • Connect changes in β-hydroxybutyrylation to gene expression patterns

  • Consider the genomic locations of β-hydroxybutyrylation changes (promoters, enhancers, gene bodies)

  • Relate findings to known metabolic adaptation pathways and stress responses

• Experimental design considerations:

  • Include appropriate time points to capture the dynamic nature of the modification

  • Consider dose-response relationships when using exogenous β-hydroxybutyrate

  • Account for potential cell type-specific differences in β-hydroxybutyrylation patterns

What are promising research areas for β-hydroxybutyryl-HIST1H2BC (K120) investigation?

Several exciting research directions exist for further investigation of β-hydroxybutyryl-HIST1H2BC (K120):

• Clinical relevance exploration:

  • Investigate the role of β-hydroxybutyrylation in metabolic disorders beyond diabetes

  • Explore potential connections to neurological conditions where ketone metabolism is altered

  • Examine β-hydroxybutyrylation patterns in cancer metabolism and potential therapeutic implications

• Molecular mechanism elucidation:

  • Identify specific "reader" proteins that recognize β-hydroxybutyrylation at K120

  • Characterize the enzymes responsible for adding and removing this modification

  • Determine the structural consequences of β-hydroxybutyrylation on nucleosome stability

• Therapeutic targeting possibilities:

  • Develop small molecules that can specifically modulate β-hydroxybutyrylation levels

  • Explore the potential of ketogenic diets or β-hydroxybutyrate supplementation in modifying epigenetic states

  • Investigate combination approaches targeting multiple histone modifications simultaneously

• Technological advancement needs:

  • Develop more specific tools for detecting and manipulating site-specific β-hydroxybutyrylation

  • Create cellular models with mutated K120 sites to assess functional significance

  • Employ cutting-edge proteomics to map the complete β-hydroxybutyrylation landscape

These research directions could significantly advance our understanding of how metabolism interfaces with epigenetic regulation and potentially lead to novel therapeutic approaches for metabolic and other diseases.