β-hydroxybutyryl-HIST1H4A (K91) Antibody

What is β-hydroxybutyryl-HIST1H4A (K91) Antibody and what cellular targets does it recognize?

β-hydroxybutyryl-HIST1H4A (K91) antibody is a polyclonal antibody derived from rabbit hosts that specifically recognizes the β-hydroxybutyrylation post-translational modification at lysine 91 on histone H4. Histone H4 serves as a core component of nucleosomes, which wrap and compact DNA into chromatin. This antibody targets a specific region containing the modified lysine 91 residue, enabling researchers to detect endogenous levels of this modification in human cells. The antibody specifically recognizes the peptide sequence around the site of β-hydroxybutyryl-Lys (91) derived from Human Histone H4, making it valuable for studying this particular epigenetic mark .

How does β-hydroxybutyrylation differ from other histone post-translational modifications?

β-hydroxybutyrylation represents a unique class of acylation modification distinct from more commonly studied marks like acetylation, methylation, and formylation. Unlike acetylation, which involves the addition of an acetyl group derived from acetyl-CoA, β-hydroxybutyrylation adds a β-hydroxybutyryl group, typically derived from the ketone body β-hydroxybutyrate (BHB). This modification has been implicated in metabolic regulation of gene expression, particularly during states of altered metabolism such as ketosis, fasting, or diabetes. The chemical structure of β-hydroxybutyryl groups contains a hydroxyl group not present in acetylation or butyrylation, allowing it to potentially form additional hydrogen bonds with interacting proteins. When comparing β-hydroxybutyrylation at K91 on histone H4 to other modifications like formylation (as seen with H4K91formyl), each creates distinct functional consequences for chromatin structure and gene regulation .

What experimental applications is β-hydroxybutyryl-HIST1H4A (K91) Antibody suitable for?

The β-hydroxybutyryl-HIST1H4A (K91) antibody has been validated for several research applications:

The antibody shows strong reactivity with human samples and may cross-react with other species with high sequence homology. It is important to note that this antibody is intended strictly for research purposes and not for diagnostic or therapeutic applications. When using for Western blotting, researchers should optimize blocking conditions (typically 5% BSA in TBST is recommended) and antibody concentration for their specific experimental setup .

How can researchers validate the specificity of β-hydroxybutyryl-HIST1H4A (K91) Antibody?

Validating antibody specificity is critical for histone modification research, especially given recent findings about cross-reactivity issues with similar antibodies. The following validation approach is recommended:

• Peptide Competition Assays: Pre-incubate the antibody with increasing concentrations of both target (β-hydroxybutyrylated K91) and non-target (unmodified, acetylated, or other modified) peptides before Western blotting to assess specific blocking.

• Mass Spectrometry Confirmation: Use immunoprecipitation (IP) followed by mass spectrometry to confirm that the antibody is enriching the intended modification. This is particularly important as recent studies have shown that some histone modification antibodies can recognize unintended modifications. For example, research on the H3K9bhb antibody revealed it recognized other modifications, including acetylations, complicating data interpretation .

• Knockout/Knockdown Controls: When feasible, use genetic approaches to eliminate the target protein or the enzymes responsible for the modification.

• Treatment Controls: Compare samples with and without β-hydroxybutyrate (BHB) treatment, as BHB exposure should increase the specific signal if the antibody is truly specific.

• Cross-Modification Testing: Test the antibody against cells treated with structurally similar compounds like butyrate or histone deacetylase inhibitors like TSA to assess cross-reactivity with similar modifications .

A comprehensive validation should include at least three of these approaches before using the antibody for chromatin immunoprecipitation (ChIP) or other genome-wide studies.

What are the optimal experimental conditions for using β-hydroxybutyryl-HIST1H4A (K91) Antibody in chromatin immunoprecipitation?

When performing chromatin immunoprecipitation (ChIP) with β-hydroxybutyryl-HIST1H4A (K91) antibody, consider the following optimization protocol:

• Crosslinking Optimization: Use 1% formaldehyde for 10 minutes at room temperature for standard crosslinking. For histone modifications like β-hydroxybutyrylation, a milder crosslinking may preserve epitope accessibility.

• Chromatin Fragmentation: Optimize sonication conditions to achieve DNA fragments of 200-500 bp. Over-sonication may destroy epitopes while under-sonication results in poor resolution.

• Antibody Amount: Begin with 2-5 μg of antibody per ChIP reaction with 25-50 μg of chromatin. Titrate if necessary.

• Blocking Reagents: Use 5% BSA in TBST for blocking to reduce background, as recommended for Western blotting with this antibody.

• Washing Stringency: Include progressively more stringent wash steps to reduce non-specific binding while preserving specific interactions.

• Controls: Always include:

  • Input control (non-immunoprecipitated chromatin)

  • IgG control (matched isotype)

  • Positive control (antibody to abundant histone mark)

  • Treatment control (cells with/without BHB treatment)

• Elution and Reversal: Carefully optimize elution conditions and crosslink reversal to maximize recovery of bound DNA without introducing bias.

Remember that for any ChIP experiment using this antibody, validation of specificity is particularly important given the concerns raised about similar histone modification antibodies .

What are common causes of non-specific signals when using β-hydroxybutyryl-HIST1H4A (K91) Antibody?

Several factors can contribute to non-specific signals when using β-hydroxybutyryl-HIST1H4A (K91) antibody:

• Cross-reactivity with Similar Modifications: Recent research has highlighted that antibodies against specific histone modifications may recognize structurally similar modifications. For example, studies have shown that H3K9bhb antibody can recognize acetylation marks when cells are treated with histone deacetylase inhibitors like TSA .

• Blocking Insufficiency: Inadequate blocking can lead to non-specific binding. Using 5% BSA in TBST is generally recommended over milk-based blockers for phospho and other PTM-specific antibodies.

• Fixation Artifacts: Excessive or improper fixation can create epitopes that might be recognized by the antibody. This is particularly relevant for immunohistochemistry or immunofluorescence applications.

• Batch-to-Batch Variation: Polyclonal antibodies like β-hydroxybutyryl-HIST1H4A (K91) antibody may show variation between production lots. Always validate new lots against previous successful experiments.

• Sample Preparation Issues: Protein degradation during sample preparation can generate fragments that cause unexpected bands. Always include protease inhibitors and maintain appropriate temperatures during preparation.

To address these issues, researchers should include additional controls, such as peptide competition assays and samples treated with and without BHB. Immunoprecipitation followed by mass spectrometry analysis can definitively identify what the antibody is actually recognizing in your experimental system .

How can I optimize Western blot protocols for β-hydroxybutyryl-HIST1H4A (K91) Antibody?

For optimal Western blot results with β-hydroxybutyryl-HIST1H4A (K91) antibody, follow these guidelines:

• Sample Preparation:

  • Extract histones using acid extraction methods that maintain post-translational modifications

  • Include deacetylase inhibitors (e.g., sodium butyrate, TSA) and phosphatase inhibitors in extraction buffers

  • Use fresh samples when possible or store at -80°C with minimal freeze-thaw cycles

• Gel and Transfer Optimization:

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

  • Consider using Triton-acid-urea (TAU) gels for better separation of modified histones

  • Transfer at lower voltage (30V) overnight at 4°C to ensure complete transfer of small histone proteins

• Antibody Incubation:

  • Start with 1:500 dilution in 5% BSA/TBST as recommended

  • Incubate overnight at 4°C with gentle rocking

  • For low abundance modifications, consider extending primary antibody incubation time

• Signal Development:

  • Use enhanced chemiluminescence (ECL) detection systems appropriate for the expected signal strength

  • Begin with short exposure times (5 seconds) and increase as needed

  • Consider using fluorescence-based secondary antibodies for more quantitative results

• Controls to Include:

  • Positive control: BHB-treated cell lysate

  • Negative control: Untreated cell lysate

  • Peptide competition control: Antibody pre-incubated with specific peptide

  • Loading control: Total H4 or other histone variant

The expected molecular weight of histone H4 is approximately 11-12 kDa. Be aware that the migration pattern may be affected by the presence of post-translational modifications .

How does β-hydroxybutyrylation at H4K91 relate to other histone marks in gene regulation?

β-hydroxybutyrylation at H4K91 represents an important but still emerging area of histone modification research. This modification exists within a complex landscape of histone marks that collectively regulate chromatin structure and gene expression. Understanding its relationship to other marks provides critical context:

• Positional Significance: Lysine 91 on histone H4 is located in the globular domain rather than the N-terminal tail where many well-studied modifications occur. This positioning may affect nucleosome stability and higher-order chromatin structure rather than directly influencing transcription factor binding.

• Metabolic Signaling: Unlike many histone modifications, β-hydroxybutyrylation serves as a direct link between cellular metabolism and epigenetic regulation. It increases during states of elevated β-hydroxybutyrate levels, such as fasting, ketogenic diet, or diabetic ketosis, suggesting a role in adapting gene expression to metabolic state.

• Co-occurrence Patterns: Research should examine whether H4K91bhb co-occurs with or is mutually exclusive with other modifications at this position, such as H4K91 acetylation or H4K91 formylation . These relationships can provide insights into the biological functions and regulatory mechanisms.

• Cross-talk with Other Modifications: Evidence from studies on other histone marks suggests that modifications can influence each other through recruitment or inhibition of modifying enzymes. Investigating whether H4K91bhb affects other modifications on the same or adjacent nucleosomes would be valuable.

• Reader Proteins: Identifying proteins that specifically recognize H4K91bhb (readers) would help elucidate its downstream effects on chromatin structure and gene expression.

How can mass spectrometry complement antibody-based detection of β-hydroxybutyryl-HIST1H4A (K91)?

Mass spectrometry (MS) provides a powerful complementary approach to antibody-based detection of histone modifications like β-hydroxybutyrylation:

A recommended integrated workflow combines immunoprecipitation using the β-hydroxybutyryl-HIST1H4A (K91) antibody followed by MS analysis of the enriched proteins to verify the target modification and identify potential cross-reactivities. This approach provides both the enrichment capabilities of antibodies and the specificity of MS identification .

What are the emerging applications of β-hydroxybutyryl-HIST1H4A (K91) Antibody in metabolic research?

β-hydroxybutyrylation represents a direct link between metabolism and epigenetic regulation, opening several promising research directions:

• Ketogenic Diet Studies: Researchers can use the β-hydroxybutyryl-HIST1H4A (K91) antibody to investigate how ketogenic diets alter gene expression through histone modifications. This may provide molecular mechanisms underlying the beneficial effects of these diets in conditions like epilepsy and neurodegenerative diseases.

• Diabetes Research: Since diabetic ketoacidosis involves elevated β-hydroxybutyrate levels, studying H4K91bhb in diabetic models could reveal how metabolic dysregulation affects gene expression in this condition.

• Aging and Caloric Restriction: Intermittent fasting and caloric restriction, which can increase ketone body production, are associated with longevity benefits. Examining H4K91bhb patterns during these interventions may help explain their epigenetic impacts.

• Exercise Physiology: During prolonged exercise, ketone body production increases. Studying how exercise-induced metabolic changes affect H4K91bhb could provide insights into exercise-induced adaptations at the epigenetic level.

• Cancer Metabolism: Cancer cells often exhibit altered metabolism. Investigating the role of H4K91bhb in cancer models might reveal connections between cancer-specific metabolic states and epigenetic dysregulation.

For these applications, it is essential to use carefully validated antibodies and complementary techniques like mass spectrometry to ensure accurate identification of the modification. The concerns raised about specificity of similar histone modification antibodies underline the importance of rigorous validation in each experimental system .

What statistical approaches are appropriate for analyzing ChIP-seq data generated with β-hydroxybutyryl-HIST1H4A (K91) Antibody?

Analysis of ChIP-seq data for histone modifications requires careful statistical consideration:

• Peak Calling Algorithms:

  • For broadly distributed modifications like many histone marks, algorithms designed for broad peaks (e.g., SICER, MACS2 with broad peak options) are more appropriate than those designed for transcription factor binding sites.

  • Consider using multiple peak callers and focusing on consensus peaks to increase confidence.

• Input Normalization:

  • Always use input control (non-immunoprecipitated chromatin) for normalization to account for biases in chromatin preparation.

  • Consider using spike-in controls (e.g., chromatin from another species) for more accurate normalization, especially when global levels of modification might change between conditions.

• Differential Binding Analysis:

  • Tools like DiffBind, MAnorm, or DESeq2 can identify regions with significant changes in modification levels between conditions.

  • Select statistical thresholds based on biological questions (e.g., more stringent thresholds for identifying high-confidence targets).

• Integration with Other Data Types:

  • Correlate H4K91bhb peaks with gene expression data (RNA-seq) to identify functional relationships.

  • Compare with other histone modification profiles to understand the chromatin context.

  • Integrate with transcription factor binding data to identify potential regulatory mechanisms.

• Addressing Antibody Specificity Concerns:

  • As indicated by research on similar antibodies like H3K9bhb, cross-reactivity can significantly impact data interpretation .

  • Consider filtering peaks based on additional criteria such as presence in BHB-treated samples but not in untreated controls.

  • Validate key findings with orthogonal approaches like mass spectrometry.

• Visualization and Reporting:

  • Use genome browsers to visualize data along with other relevant tracks.

  • Report peak distributions relative to genomic features (promoters, enhancers, gene bodies).

  • Include quality metrics such as fragment length distribution, library complexity, and enrichment over background.

These approaches help ensure robust interpretation of ChIP-seq data while accounting for the potential limitations of antibody-based detection methods .