Butyrly-HIST1H3A (K9) Antibody

What is Butyryl-HIST1H3A (K9) Antibody and what does it detect?

Butyryl-HIST1H3A (K9) Antibody is a polyclonal antibody raised in rabbits that targets histone H3.1 (HIST1H3A) specifically modified with butyrylation at lysine 9 (K9). The antibody was developed to detect this post-translational modification (PTM) that occurs on histone proteins and is associated with chromatin regulation and gene expression. The immunogen used to generate this antibody is a synthetic peptide sequence surrounding the butyrylated lysine 9 site derived from Human Histone H3.1 . The antibody is designed to recognize the butyryl group attached to the epsilon-amino group of lysine 9 on histone H3.1, which is an important epigenetic mark.

What are the validated applications for Butyryl-HIST1H3A (K9) Antibody?

The Butyryl-HIST1H3A (K9) Antibody has been validated for multiple experimental applications in epigenetic research:

These applications enable researchers to detect and quantify butyrylated histones in various experimental contexts . For optimal results, the appropriate dilution should be determined empirically for each specific experimental system.

How should Butyryl-HIST1H3A (K9) Antibody samples be prepared and stored?

For optimal antibody performance and longevity, Butyryl-HIST1H3A (K9) Antibody should be stored following these guidelines:

• The antibody is supplied in liquid form in a buffer containing 0.01 M PBS, pH 7.4, 0.03% Proclin-300, and 50% glycerol .

• Upon receipt, aliquot the antibody to avoid repeated freeze-thaw cycles, which can degrade antibody quality.

• Store aliquots at -20°C for long-term preservation .

• When preparing for experiments, thaw aliquots at room temperature and keep on ice during use.

• Avoid exposing the antibody to high temperatures or direct sunlight.

Following these storage protocols will help maintain antibody specificity and sensitivity for experimental applications.

What is the difference between butyrylation and β-hydroxybutyrylation on histone H3?

Though structurally similar, these modifications have distinct biochemical properties and potentially different functional implications:

The critical distinction is that β-hydroxybutyrylation contains an additional hydroxyl group compared to butyrylation. This structural difference affects how these modifications are recognized by reader proteins and potentially their functional consequences on chromatin structure and gene expression .

What specificity issues have been identified with commercial H3K9bhb antibodies?

Recent studies have revealed significant specificity concerns with commercially available H3K9bhb antibodies:

Researchers found that widely used antibodies against β-hydroxybutyrylated lysine 9 on histone H3 (H3K9bhb) recognize multiple histone modifications beyond their intended target . Key findings include:

• Both monoclonal and polyclonal H3K9bhb antibodies produced signals in cells treated with butyrate or TSA (trichostatin A, a histone deacetylase inhibitor), despite these treatments not increasing intracellular BHB levels .

• Mass spectrometry analysis of immunoprecipitated samples revealed that while H3K9bhb-containing peptides were enriched in BHB-treated samples (13.99% of identified peptides), they were minimally present in butyrate-treated samples (1.74%) .

• Antibody cross-reactivity was documented with other modifications including acetylation, particularly H3K9ac, which was strongly enriched in butyrate-treated samples pulled down with the H3K9bhb antibody .

These findings indicate that caution must be exercised when interpreting results obtained using these antibodies, particularly in ChIP experiments designed to identify H3K9bhb-regulated genes .

How should researchers design controls to validate H3K9bhb antibody specificity?

To ensure experimental validity when using Butyryl-HIST1H3A (K9) antibodies, implement these critical controls:

• Peptide competition assay: Pre-incubate the antibody with synthetic peptides containing either butyrylated K9, β-hydroxybutyrylated K9, acetylated K9, or unmodified K9 to assess specific binding inhibition.

• Multiple modification treatments: Compare signals from cells treated with:

  • β-hydroxybutyrate (BHB) - expected to increase true H3K9bhb

  • Butyrate - increases butyrylation and acetylation

  • TSA (histone deacetylase inhibitor) - increases acetylation

  • Untreated controls

• Orthogonal detection methods: Validate antibody-based findings with mass spectrometry to directly identify and quantify histone modifications .

• Knockout/knockdown controls: Use cells with mutations in enzymes responsible for installing or removing these modifications.

The differential response to these treatments, as shown in published research, can help distinguish true positives from cross-reactivity .

What methodological approach is recommended for ChIP experiments using H3K9bhb antibodies?

When performing ChIP experiments with H3K9bhb antibodies, researchers should implement this optimized protocol to account for known specificity issues:

• Experimental design:

  • Include parallel ChIP experiments with H3K9ac and other potentially cross-reactive modification antibodies

  • Use both BHB-treated and untreated cells as positive and negative controls

  • Consider sequential ChIP (re-ChIP) to improve specificity

• Validation strategy:

  • Perform western blot analysis prior to ChIP to confirm the antibody detects increased signal in BHB-treated cells

  • Include input controls and IgG controls for background binding

  • Follow up with targeted mass spectrometry of ChIP-enriched regions

• Data interpretation:

  • Compare ChIP-seq peaks with known genomic distributions of other histone marks

  • Be cautious when interpreting results from butyrate or HDAC inhibitor treatments

  • Consider that signal may represent a combination of modifications rather than H3K9bhb specifically

This approach acknowledges the established cross-reactivity of H3K9bhb antibodies while maximizing experimental value.

How can mass spectrometry validate histone butyrylation detection?

Mass spectrometry provides critical validation for antibody-based detection of histone butyrylation through these methodological steps:

• Sample preparation:

  • Perform antibody immunoprecipitation from cells treated with BHB, butyrate, or control conditions

  • Extract histones using acid extraction followed by propionylation of unmodified lysines

  • Digest with trypsin to generate peptides containing the modification site

• MS analysis approach:

  • Use high-resolution LC-MS/MS to identify modified peptides

  • Employ parallel reaction monitoring for targeted analysis of specific modifications

  • Analyze fragment ions that distinguish between similar modifications (butyrylation vs. β-hydroxybutyrylation)

• Data interpretation:

  • Quantify the percentage of peptides containing specific modifications:

    • In BHB-treated samples: 13.99% of H3 peptides contained Kbhb modifications

    • In butyrate-treated samples: only 1.74% contained Kbhb modifications

  • Compare the prevalence of H3K9bhb vs. H3K9ac in samples pulled down with H3K9bhb antibody

This approach revealed that commercial H3K9bhb antibodies recognize multiple PTMs, demonstrating why orthogonal validation is essential .

What are the implications of antibody cross-reactivity for published research on H3K9bhb?

The recently documented cross-reactivity of H3K9bhb antibodies has significant implications for published literature:

This situation highlights the importance of stringent validation for antibodies targeting post-translational modifications with similar chemical structures .

What factors might cause high background or non-specific signals when using Butyryl-HIST1H3A (K9) Antibody?

Several factors can contribute to non-specific signals when using Butyryl-HIST1H3A (K9) Antibody:

• Cross-reactivity with similar modifications: The antibody may recognize acetylation, particularly H3K9ac, or other acylations at the same position. This is especially problematic in cells with high levels of histone acetylation (e.g., after HDAC inhibitor treatment) .

• Blocking inefficiency: Insufficient blocking can lead to non-specific binding. Use 5% BSA or 10% normal serum from the species of the secondary antibody .

• Antibody concentration: Excessive antibody concentration increases background. Titrate the antibody to determine optimal dilution (starting with 1/2000 for WB and 1/50 for ICC/IF as recommended) .

• Fixation artifacts: Over-fixation can expose epitopes that promote non-specific binding. Optimize fixation conditions (e.g., 4% formaldehyde for 10-15 minutes for IF/ICC) .

• Secondary antibody cross-reactivity: Ensure the secondary antibody is highly cross-adsorbed against potential interfering species.

These factors highlight the importance of including proper controls and validation steps in experimental design .

How do different cellular treatments affect the detection pattern of histone butyrylation?

Different treatments produce distinct patterns of histone modifications that impact Butyryl-HIST1H3A (K9) Antibody binding:

These differential responses underline why researchers should include multiple treatment conditions as controls and confirm antibody-based findings with mass spectrometry whenever possible .

What approaches can improve the specificity of antibodies targeting histone butyrylation?

To address current specificity limitations in butyrylation antibodies, several approaches show promise:

• Structural optimization: Design immunogens that emphasize the unique structural features distinguishing butyrylation from similar modifications.

• Negative selection strategies: Deplete antibody preparations using affinity columns containing cross-reactive epitopes (e.g., acetylated histones).

• Combinatorial recognition: Develop antibodies that recognize both the modification and surrounding sequence context unique to specific histone variants.

• Validation standards: Establish industry-wide validation protocols requiring demonstration of specificity against closely related modifications.

• Alternative detection technologies: Develop aptamer or nanobody-based detection reagents that may offer improved specificity.

The recognized limitations of current H3K9bhb antibodies underscore the importance of developing next-generation reagents with improved specificity profiles .

What is the functional significance of histone butyrylation in comparison to other acylations?

The biological functions of histone butyrylation are still being elucidated, but emerging research suggests distinctive roles:

• Chromatin regulation: Like acetylation, butyrylation neutralizes the positive charge of lysine residues, potentially weakening histone-DNA interactions and promoting an open chromatin state conducive to transcription.

• Metabolic sensing: Butyrylation may serve as a mechanism linking cellular metabolism to gene regulation, similar to how β-hydroxybutyrylation responds to ketogenic states.

• Reader protein interactions: The larger butyryl group likely creates a distinct binding surface that may be recognized by specific reader proteins different from those recognizing acetylation.

• Enzymatic regulation: Histone deacetylases (HDACs) can remove butyryl groups, but with different kinetics than acetyl groups, potentially creating different dynamics of regulation.

Future research using improved antibodies and orthogonal detection methods will be essential to distinguish the unique functions of butyrylation from other similar modifications .