β-hydroxybutyryl-HIST1H2BC (K20) Antibody

What is the β-hydroxybutyryl-HIST1H2BC (K20) Antibody and what does it detect?

The β-hydroxybutyryl-HIST1H2BC (K20) Antibody is a polyclonal antibody raised in rabbits that specifically recognizes the β-hydroxybutyrylation modification at lysine 20 of histone H2B type 1-C/E/F/G/I. This antibody detects a relatively recently discovered histone post-translational modification that has significant implications for cellular metabolism and gene regulation . The antibody specifically targets the peptide sequence around the site of β-hydroxybutyryl-Lys (20) derived from Human Histone H2B type 1-C/E/F/G/I .

β-hydroxybutyrylation (Kbhb) is one of at least 44 identified histone Kbhb sites, a number comparable to known histone acetylation sites . This particular modification is part of a broader histone code that regulates DNA accessibility to cellular machinery, affecting transcription regulation, DNA repair, DNA replication, and chromosomal stability .

What are the validated applications for this antibody?

The β-hydroxybutyryl-HIST1H2BC (K20) Antibody has been validated for multiple research applications with specific recommended dilutions for optimal results:

The antibody has been specifically tested and validated with human samples, showing high specificity for β-hydroxybutyrylation at the K20 position of histone H2B . For Western blotting applications, the antibody has been successfully used to detect the modification in multiple cell types, including 293 whole cell lysate and A549 whole cell lysate .

How does β-hydroxybutyrylation differ from other histone modifications?

β-hydroxybutyrylation (Kbhb) represents a distinct histone modification with unique metabolic connections compared to other well-studied modifications:

• Metabolic origin: Unlike acetylation (which uses acetyl-CoA), β-hydroxybutyrylation specifically utilizes β-hydroxybutyryl-CoA as its cofactor, which is derived from β-hydroxybutyrate, a ketone body that increases during fasting or diabetic ketoacidosis .

• Regulation mechanism: Histone Kbhb levels are directly regulated by cellular β-hydroxybutyrate concentrations, creating a direct link between metabolic state and gene regulation. This differs from many other histone modifications that respond to complex signaling cascades .

• Response pattern: Research has demonstrated that histone Kbhb levels can be dramatically induced in response to elevated β-hydroxybutyrate, while histone acetylation levels remain relatively unchanged under the same conditions .

• Genomic distribution: ChIP-seq analysis has shown that histone Kbhb is specifically enriched in active gene promoters, particularly those associated with starvation-responsive metabolic pathways during fasting conditions .

What controls should be included when using this antibody?

When designing experiments using the β-hydroxybutyryl-HIST1H2BC (K20) Antibody, the following controls should be incorporated to ensure reliable and interpretable results:

• Negative controls:

  • Isotype control (rabbit IgG at matching concentration)

  • Untreated cells/tissues (with low β-hydroxybutyrate levels) to establish baseline levels

  • Secondary antibody-only control to assess non-specific binding

• Positive controls:

  • Cells treated with sodium β-hydroxybutyrate (5-10 mM) to induce β-hydroxybutyrylation

  • Samples from fasted animals or diabetic models known to have elevated β-hydroxybutyrate levels

• Specificity controls:

  • Peptide competition assay using the immunogenic peptide to confirm binding specificity

  • Parallel analysis with antibodies against different histone modifications (e.g., H3K9ac) to compare modification patterns

• Loading controls:

  • Total H2B antibody to normalize for histone content variation

  • Housekeeping proteins (for whole cell lysates) to ensure equal loading

What is the biological significance of histone H2B K20 β-hydroxybutyrylation?

Histone H2B K20 β-hydroxybutyrylation represents a critical interface between cellular metabolism and gene regulation. This modification is:

• Metabolically responsive: Levels of H2B K20bhb increase dramatically in response to elevated β-hydroxybutyrate, which occurs naturally during prolonged fasting or in pathological conditions like diabetic ketoacidosis .

• Evolutionarily conserved: The modification has been detected across diverse eukaryotic species including yeast, Drosophila, mice, and humans, suggesting fundamental biological importance .

• Gene regulation mediator: H2B K20bhb, along with other Kbhb marks, is enriched in active gene promoters and is associated with upregulation of genes involved in starvation-responsive metabolic pathways .

• Chromatin structure modulator: As a core nucleosomal component, modified H2B contributes to regulating DNA accessibility to transcriptional machinery, affecting fundamental processes such as transcription regulation, DNA repair, and replication .

• Potential therapeutic target: Understanding H2B K20bhb may provide insights into metabolic disorders and potential therapeutic approaches targeting the interface between metabolism and gene regulation.

How can I design ChIP-seq experiments to study genome-wide distribution of H2B K20bhb?

Designing effective ChIP-seq experiments to map the genome-wide distribution of H2B K20bhb requires careful planning and specific methodological considerations:

• Experimental design considerations:

  • Include appropriate metabolic conditions: Compare standard culture, β-hydroxybutyrate treatment (10mM), and physiological models like fasting or diabetic ketoacidosis

  • Perform parallel ChIP-seq with other histone marks (H3K4me3, H3K27ac) to correlate with active transcription

  • Include RNA-seq analysis of the same samples to correlate H2B K20bhb enrichment with gene expression changes

• Optimized ChIP protocol for Kbhb detection:

  • Crosslinking: Standard 1% formaldehyde fixation for 10 minutes at room temperature

  • Chromatin preparation: Sonication to 200-500bp fragments

  • Immunoprecipitation: Use 5μg of β-hydroxybutyryl-HIST1H2BC (K20) antibody per IP reaction

  • Include IgG and total H2B ChIP controls for normalization

  • Validate antibody specificity using peptide competition prior to sequencing

• Data analysis pipeline:

  • Map reads to appropriate reference genome (hg38 for human studies)

  • Call peaks using MACS2 with appropriate settings for histone modifications

  • Normalize signal to input and IgG controls

  • Perform differential binding analysis between conditions

  • Correlate with gene expression data from RNA-seq

  • Conduct motif enrichment analysis at Kbhb-marked regions

  • Perform gene ontology and pathway analysis of genes associated with H2B K20bhb-enriched regions

• Validation strategies:

  • Confirm key findings using ChIP-qPCR at selected loci

  • Validate with complementary techniques like CUT&RUN or CUT&Tag

  • Perform functional studies with genes showing differential Kbhb marking

What is the relationship between β-hydroxybutyrylation and other histone marks in metabolic regulation?

The interplay between histone β-hydroxybutyrylation and other histone modifications forms a complex regulatory network that integrates metabolic signals with gene expression:

How can I troubleshoot specificity issues with the β-hydroxybutyryl-HIST1H2BC (K20) Antibody?

When facing specificity challenges with the β-hydroxybutyryl-HIST1H2BC (K20) Antibody, systematic troubleshooting can help resolve these issues:

• Validating antibody specificity:

  • Perform dot blot assays with synthetic peptides containing K20bhb versus unmodified, acetylated, or other acylated forms of the H2B K20 peptide

  • Conduct peptide competition assays using increasing concentrations of the immunogenic peptide to demonstrate specific blocking

  • Test antibody reactivity in cells or tissues with naturally low β-hydroxybutyrate levels compared to those with elevated levels

• Addressing cross-reactivity issues:

  • β-hydroxybutyrylation (+86.0368 Da) can be confused with other modifications of similar mass

  • Verify specificity against other related acylations like β-hydroxyisobutyrylation, 2-hydroxybutyrylation, 2-hydroxyisobutyrylation, or 4-hydroxybutyrylation by testing with synthetic peptides containing these modifications

  • Use high-resolution mass spectrometry to confirm the exact modification detected

• Optimizing experimental conditions:

  • Antibody dilution: Test a range of dilutions (beyond the recommended range) to find optimal signal-to-noise ratio

  • Blocking conditions: Evaluate different blocking agents (BSA, milk, serum) to reduce background

  • Incubation time and temperature: Adjust to improve specific binding and reduce non-specific interactions

  • Washing stringency: Increase number and duration of washes to reduce background

• Confirming results with complementary approaches:

  • Use isotopic labeling with 13C-labeled β-hydroxybutyrate to track specific incorporation into histones

  • Employ mass spectrometry to verify the exact modification at H2B K20

  • Use site-directed mutagenesis (K20R) to confirm antibody specificity in cellular systems

What are the methodological considerations for studying β-hydroxybutyrylation in metabolic disease models?

When investigating histone β-hydroxybutyrylation in metabolic disease models, several methodological considerations are critical:

• Model selection and validation:

  • Diabetic ketoacidosis models: Streptozotocin (STZ)-induced Type 1 diabetes models show 10-fold elevations in β-hydroxybutyrate levels and significant increases in histone Kbhb

  • Fasting models: Prolonged fasting (typically 24-48 hours in mice) induces elevated β-hydroxybutyrate and histone Kbhb

  • Cell culture models: Treatment with sodium β-hydroxybutyrate (5-10mM) in cultured cells simulates ketotic states

  • Always confirm metabolic parameters: Measure blood glucose and β-hydroxybutyrate levels to validate model status

• Tissue and cellular considerations:

  • Tissue selection: Liver shows pronounced Kbhb changes in metabolic stress; other metabolically active tissues (muscle, adipose, kidney) may show tissue-specific responses

  • Cell type heterogeneity: Consider cell type-specific effects using techniques like single-cell approaches or cell sorting

  • Temporal dynamics: Design time-course studies to capture acute versus chronic adaptations

• Experimental controls and comparisons:

  • Within-subject controls: Use paired tissue samples where possible

  • Between-group normalization: Ensure groups are matched for age, sex, genetic background

  • Intervention controls: Include non-ketogenic interventions to distinguish Kbhb-specific effects

  • Recovery groups: Include groups where metabolic perturbation is resolved to assess reversibility

• Integrated analysis approaches:

  • Combine histone Kbhb profiling with:

    • Metabolomics to measure β-hydroxybutyrate and related metabolites

    • Transcriptomics to correlate with gene expression changes

    • Enzyme activity assays for writers/erasers of Kbhb

    • Functional assays relevant to the metabolic disease being studied

• Analytical considerations:

  • Ensure preservation of labile histone modifications during sample preparation

  • Consider both global and site-specific Kbhb quantification

  • Design appropriate statistical approaches for multiple comparisons

  • Validate key findings with orthogonal methods

How can isotopic labeling be used to track de novo β-hydroxybutyrylation in cellular systems?

Isotopic labeling provides powerful approaches for tracking the dynamics and specificity of histone β-hydroxybutyrylation in cellular systems:

• Experimental design using isotopic labeling:

  • Selection of isotope: 13C-labeled sodium β-hydroxybutyrate is effective for tracking incorporation into histones

  • Labeling protocol: Cells are treated with isotopically labeled β-hydroxybutyrate (typically 10mM) for varying time periods

  • Control conditions: Include unlabeled β-hydroxybutyrate treatment and untreated controls

  • Dose-dependency: Test multiple concentrations of labeled compound (1-20mM) to establish relationship between substrate availability and histone modification

• Sample preparation and analysis:

  • Histone extraction: Acid extraction methods preserve post-translational modifications

  • Enzymatic digestion: Trypsin digestion generates peptides suitable for MS analysis

  • Mass spectrometry approach: High-resolution LC-MS/MS to detect mass shifts corresponding to labeled β-hydroxybutyrylation

  • Data analysis: Search for peptides with +2Da mass shift (for 13C2-labeled bhb) compared to unlabeled modification (+86.0368 Da)

• Tracking metabolic conversion pathways:

  • Monitor conversion of labeled β-hydroxybutyrate to β-hydroxybutyryl-CoA using metabolomics approaches

  • Quantify dose-dependent increases in labeled bhb-CoA to establish precursor-product relationship

  • Examine potential metabolic interconversions to other acyl-CoA species

• Validation and functional interpretation:

  • Confirm identification by comparing fragmentation patterns of labeled peptides with synthetic standards

  • Perform pulse-chase experiments to determine turnover rates of the modification

  • Combine with ChIP-seq using β-hydroxybutyryl-HIST1H2BC (K20) antibody to map genomic distribution of newly deposited marks

  • Correlate with RNA-seq to determine functional consequences of de novo β-hydroxybutyrylation