Butyrly-HIST1H4A (K12) Antibody

What is Butyryl-HIST1H4A (K12) and what are its validated applications?

Butyryl-HIST1H4A (K12) refers to the butyrylation (a post-translational modification) at lysine 12 of histone H4, a core histone protein crucial for chromatin structure and function. The Butyryl-HIST1H4A (K12) Polyclonal Antibody has been extensively validated for multiple applications including ELISA, Western blot (WB), immunocytochemistry (ICC), immunofluorescence (IF), immunoprecipitation (IP), and chromatin immunoprecipitation (ChIP) . This antibody specifically recognizes the butyrylation modification at the K12 position of human histone H4, making it valuable for epigenetic research involving post-translational histone modifications .

How does butyrylation differ from acetylation at the K12 position of histone H4?

While both butyrylation and acetylation are acylation modifications that occur on lysine residues, they differ in their chemical structures and functional implications. Butyrylation involves the addition of a four-carbon butyryl group (CH₃CH₂CH₂CO-), whereas acetylation adds a two-carbon acetyl group (CH₃CO-) . This structural difference contributes to distinct functional outcomes in chromatin regulation. Research indicates that butyrylation and acetylation can compete for the same lysine residues on histone H4, including K12, creating a dynamic interplay that affects protein-histone interactions . Unlike acetylation, which is well known to generally promote gene expression, butyrylation may have more specialized regulatory functions that are still being characterized .

What controls should be included when using Butyryl-HIST1H4A (K12) antibody in experimental design?

When designing experiments with the Butyryl-HIST1H4A (K12) antibody, researchers should implement several critical controls:

• Peptide Competition Assay: Pre-incubate the antibody with synthetic peptides containing butyrylated K12 to verify specificity.

• Modification-Free Control: Include unmodified histone H4 samples to confirm modification-dependent recognition.

• Cross-Reactivity Controls: Test against samples with other modifications at K12 (acetylation, methylation) and butyrylation at other lysine positions to ensure specificity .

• Genetic Controls: When possible, use cells with mutations at K12 that prevent butyrylation to validate antibody specificity .

• Antibody Dilution Series: Perform titration experiments to determine optimal antibody concentration for each application .

These controls help establish reliable experimental conditions and validate the specificity of the observed signals, particularly important given the similar structure of different acylation modifications.

How can I verify the specificity of Butyryl-HIST1H4A (K12) antibody against other histone modifications?

Verifying antibody specificity requires a multi-faceted approach:

• ELISA Testing: Use synthetic peptides with defined modifications to test cross-reactivity. Create a panel including:

  • Butyrylated H4K12 peptides (target)

  • Acetylated H4K12 peptides (similar modification)

  • Butyrylation at nearby residues (K5, K8, K16)

  • Unmodified peptides as negative controls

• Immunoblotting with Recombinant Proteins: Express recombinant histone H4 with specific modifications or substitution mutations (K12A, K12R) to test reactivity patterns .

• Dot Blot Analysis: Apply decreasing concentrations of modified peptides to membranes to determine detection sensitivity and specificity thresholds.

• Surface Plasmon Resonance (SPR): Measure antibody binding affinity to different modified peptides to quantitatively assess specificity .

Analysis from referenced studies suggests that high-quality antibodies like the Butyryl-HIST1H4A (K12) demonstrate minimal cross-reactivity with acetylated counterparts when properly validated .

How can I optimize ChIP-seq protocols specifically for Butyryl-HIST1H4A (K12) to distinguish it from acetylation marks?

Optimizing ChIP-seq for Butyryl-HIST1H4A (K12) requires specialized approaches to distinguish it from acetylation marks:

• Cross-linking Optimization: Butyrylation may require different cross-linking conditions than acetylation. Test a titration of formaldehyde concentrations (0.5-2%) and fixation times (5-20 minutes) to preserve the butyrylation mark without creating excessive cross-links .

• Sonication Parameters: Optimize chromatin fragmentation to 200-500bp fragments, as butyrylated regions may have different chromatin compaction properties than acetylated regions.

• Blocking and Washing Stringency: Increase blocking with BSA (3-5%) to reduce background and use more stringent wash conditions (higher salt concentrations in later washes) to eliminate weak cross-reactivity with acetylation .

• Spike-in Controls: Add exogenous DNA with known butyrylation patterns as an internal control for normalization between samples.

• Sequential ChIP Approach: Consider performing sequential ChIP (re-ChIP) with acetylation-specific antibodies followed by butyrylation-specific antibodies to identify regions with exclusive or overlapping modifications .

• Antibody Validation by Mass Spectrometry: Confirm the specificity of immunoprecipitated material using mass spectrometry analysis of a subset of samples to verify enrichment of butyrylated versus acetylated peptides .

Studies have shown that careful optimization can achieve comparable efficiency between butyrylation and acetylation ChIP-seq experiments, with antibodies showing similar ranges of affinity as measured by SPR .

What is the functional significance of competing histone H4 K12 butyrylation versus acetylation in gene regulation?

The functional interplay between H4K12 butyrylation and acetylation represents a sophisticated regulatory mechanism:

• Protein Interaction Dynamics: Butyrylation at H4K12 alters the binding affinity of chromatin-associated proteins compared to acetylation. For example, studies with the bromodomain protein Brdt show that while it can bind acetylated H4, butyrylation at specific lysines (particularly K5) inhibits this interaction . Similar differential interactions likely exist for H4K12 modifications.

• Transcriptional Regulation: H4K12 acetylation is associated with transcriptionally active regions, particularly around transcription start sites (TSSs). H4K12 butyrylation appears to have more specialized and context-dependent effects on gene expression, potentially functioning in developmental regulation or cellular stress responses .

• Cell Cycle Dependence: H4K12 modifications undergo dynamic changes during the cell cycle. The competing nature of butyrylation versus acetylation may provide a mechanism for rapid transitions between transcriptional states during cell cycle progression .

• Tissue-Specific Patterns: Research suggests that butyrylation patterns show greater tissue specificity than acetylation, with particularly important roles in specialized processes like spermatogenesis .

The current evidence indicates that butyrylation is not merely a redundant modification to acetylation but represents a distinct regulatory signal with unique downstream effects on chromatin function and gene expression .

How can I analyze the genome-wide distribution patterns of Butyryl-HIST1H4A (K12) in relation to other histone modifications?

Comprehensive genome-wide analysis of Butyryl-HIST1H4A (K12) distribution requires integrated approaches:

• Comparative ChIP-seq Analysis: Perform parallel ChIP-seq experiments for H4K12bu, H4K12ac, and other relevant modifications (H4K5bu, H4K8bu, H4K5ac, H4K8ac) using antibodies with validated and similar affinities .

• Integrated Data Analysis Pipeline:

  • Normalize signal using spike-in controls

  • Generate heatmaps and metaplots around genomic features (TSSs, enhancers)

  • Perform correlation analysis between different modifications

  • Use machine learning approaches to identify patterns of co-occurrence or mutual exclusivity

• Integration with Transcriptomic Data: Correlate modification patterns with RNA-seq data to identify the relationships between specific modifications and transcriptional activity.

• Quantitative Mass Spectrometry: Complement ChIP-seq with mass spectrometry-based approaches to determine absolute levels of different modifications and their co-occurrence on the same histone tails.

• High-Resolution Techniques: Consider CUT&RUN or CUT&Tag methods as alternatives to traditional ChIP-seq for improved signal-to-noise ratio when analyzing butyrylation marks.

Research has demonstrated that these integrated approaches can reveal distinct distribution patterns of butyrylation versus acetylation at specific genomic elements, providing insights into their specialized functions .

What methodological approaches can address the cross-reactivity challenges when studying closely related histone modifications?

Addressing cross-reactivity challenges requires sophisticated methodological solutions:

• Combinatorial Epitope Analysis: Use antibodies recognizing different epitope combinations to distinguish specific modification patterns. For example, some antibodies can detect H4K5ac only when K8 is unacetylated, allowing identification of specific modification states .

• Quantitative Mass Spectrometry Validation:

  • Perform parallel analysis of ChIP-ed material by mass spectrometry

  • Use stable isotope labeling to quantify relative abundances of different modifications

  • Implement middle-down MS approaches to analyze co-occurrence of modifications on the same histone tail

• Generation of Highly Specific Antibodies: Consider developing monoclonal antibodies with enhanced specificity through careful immunization and screening strategies .

• Orthogonal Validation: Compare results from antibody-based methods with genetic approaches:

  • Use site-specific histone mutants (K-to-R or K-to-Q) where feasible

  • Employ enzyme inhibitors to modulate specific modifications

  • Utilize CRISPR-based approaches to tag endogenous histones for alternative detection methods

• Controlled Competition Assays: Develop structured competition assays where antibodies are pre-incubated with varying concentrations of differentially modified peptides to mathematically model and correct for cross-reactivity.

These approaches have been successfully implemented in studies comparing acetylation and butyrylation patterns, revealing the distinct biological roles of these similar but functionally divergent modifications .

What are the most common causes of false positive/negative results when using Butyryl-HIST1H4A (K12) antibody?

False results with Butyryl-HIST1H4A (K12) antibody can stem from several sources that require methodical troubleshooting:

False Positives:

• Cross-reactivity with acetylation: The structural similarity between butyryl and acetyl groups can lead to recognition of H4K12ac, especially at high antibody concentrations .

• Non-specific binding to other butyrylated lysines: The antibody may detect butyrylation at nearby lysines (K5, K8, K16) if not sufficiently specific.

• Excessive antibody concentration: Using too much antibody increases background binding to non-target epitopes.

• Inadequate blocking: Insufficient blocking can lead to non-specific antibody adherence to the experimental matrix.

False Negatives:

• Epitope masking: Protein-protein interactions or adjacent modifications may block antibody access to the butyrylated K12.

• Modification instability: Butyrylation may be more labile under certain experimental conditions than acetylation, leading to modification loss during sample processing.

• Competitive displacement: In samples with high levels of both acetylation and butyrylation, competitive binding dynamics may reduce detection efficiency.

• Fixation artifacts: Excessive cross-linking can mask epitopes, particularly problematic for butyrylation which involves a larger chemical group than acetylation.

To minimize these issues, researchers should implement robust controls, carefully titrate antibody concentrations, and validate results with orthogonal methods such as mass spectrometry verification of immunoprecipitated material .

How can I properly interpret ChIP-seq data when comparing histone acetylation versus butyrylation patterns?

Interpreting comparative ChIP-seq data for acetylation versus butyrylation requires careful analytical considerations:

• Normalization Strategies:

  • Use spike-in controls with known amounts of exogenous chromatin

  • Normalize to regions where modification levels are expected to remain constant

  • Employ quantile normalization only when appropriate based on global distribution patterns

• Antibody Efficiency Correction:

  • Measure and account for differences in antibody affinities using SPR data

  • Develop correction factors based on known standard peptides to adjust signal intensities

• Peak Calling Considerations:

  • Use matched input controls for each experiment

  • Consider broader peaks for butyrylation if it shows different distribution patterns

  • Implement dual-threshold approaches to capture both strong and weak enrichment regions

• Analytical Framework for Comparison:

  • Analyze absolute enrichment at defined genomic elements

  • Examine relative enrichment patterns between modifications

  • Consider co-occurrence or mutual exclusivity of marks

• Biological Context Integration:

  • Correlate with transcriptional activity data

  • Analyze in the context of cell cycle phase information

  • Consider metabolic state of cells which may affect butyryl-CoA availability

Research indicates that butyrylation and acetylation show distinct genomic distribution patterns, with butyrylation potentially marking specialized regulatory regions or states compared to the more generally transcription-associated acetylation marks .

What methodological adaptations are needed when analyzing Butyryl-HIST1H4A (K12) in different cell types or experimental conditions?

Different cellular contexts require methodological adaptations when studying H4K12 butyrylation:

• Cell Type-Specific Considerations:

  • Dividing vs. Non-dividing Cells: Adjust chromatin preparation protocols based on nuclear compaction differences

  • Primary vs. Cell Lines: Primary cells may require gentler fixation conditions to preserve modifications

  • Tissue-Specific Cells: Consider specialized extraction buffers for cells with unique nuclear properties (e.g., neurons, spermatocytes)

• Metabolic State Adjustments:

  • Butyrylation depends on butyryl-CoA availability, which varies with metabolic conditions

  • Consider pre-treatment with butyryl-CoA precursors or HDAC inhibitors in low-butyrylation states

  • Monitor cellular metabolic parameters alongside modification analysis

• Fixation Protocol Modifications:

  • Dividing Cells: Use shorter fixation times (5-10 minutes) to avoid over-crosslinking

  • Tissues: Consider dual crosslinking protocols (formaldehyde + disuccinimidyl glutarate)

  • Sensitive Applications: Test alternative fixatives (e.g., ethylene glycol bis-succinimidyl succinate)

• Extraction and Immunoprecipitation Adjustments:

  • High-Fat Content Tissues: Include additional delipidation steps

  • Protein-Rich Samples: Increase protease inhibitor concentration

  • Tissues with High Endogenous Biotin: Include avidin pre-clearing steps for streptavidin-based protocols

• Analysis Pipeline Adaptations:

  • Tissue-Specific Reference Maps: Use appropriate tissue-matched controls

  • Developmental Studies: Implement time-series analytical approaches

  • Stress Response Analysis: Consider rapid kinetics and transient modification patterns

Research shows that butyrylation patterns can be highly context-dependent, with particular enrichment in specialized cell types like spermatocytes compared to somatic cells, necessitating these methodological adaptations .

How does the interplay between Butyryl-HIST1H4A (K12) and other histone modifications contribute to chromatin regulation?

The interplay between H4K12 butyrylation and other histone modifications creates a sophisticated regulatory network:

• Modification Crosstalk Mechanisms:

  • Sequential Modification Patterns: Evidence suggests H4K12 butyrylation may work sequentially with other modifications, where one modification serves as a prerequisite for another

  • Antagonistic Relationships: Butyrylation at H4K12 may prevent or displace other modifications at the same residue (acetylation, methylation)

  • Synergistic Effects: Certain modification combinations including H4K12bu may cooperatively enhance or suppress specific chromatin functions

• Reader Protein Dynamics:

  • Butyrylation creates a distinct binding surface compared to acetylation

  • Bromodomain-containing proteins show differential binding to acetylated versus butyrylated residues

  • Some reader proteins may recognize specific patterns involving H4K12bu in combination with other modifications

• Functional Outcomes in Chromatin Structure:

  • Butyrylation may destabilize nucleosome-DNA interactions differently than acetylation

  • The presence of H4K12bu within modification patterns can influence higher-order chromatin structures

  • Combined modification states including H4K12bu contribute to specialized chromatin domains

• Evolutionary Perspectives:

  • The conservation of lysine butyrylation sites suggests functional importance

  • Different species show varying patterns of acylation interplay, reflecting evolutionary adaptation

Research indicates that the Brdt protein, which typically binds acetylated histones, shows distinctly different binding patterns with butyrylated histones, suggesting a specialized regulatory mechanism where butyrylation can inhibit protein interactions that would otherwise occur with acetylated histones .

What role does Butyryl-HIST1H4A (K12) play in cellular differentiation and development?

Emerging research indicates specialized roles for H4K12 butyrylation in developmental processes:

• Developmental Dynamics:

  • Butyrylation patterns show significant changes during cellular differentiation

  • Specific developmental transitions feature shifts between acetylation and butyrylation at H4K12

  • Temporal regulation of butyrylation may help establish cell fate decisions

• Tissue-Specific Functions:

  • Spermatogenesis: H4K12 butyrylation appears particularly important during male germ cell development, with distinct distribution patterns compared to somatic cells

  • Neural Development: Preliminary evidence suggests roles in neuronal maturation

  • Embryonic Development: Dynamic regulation during early developmental stages

• Mechanistic Contributions:

  • Gene Poising: Butyrylation may mark developmental genes for later activation

  • Chromatin Remodeling: Facilitates developmental transitions requiring dramatic chromatin restructuring

  • Epigenetic Memory: May contribute to stable inheritance of developmental decisions

• Metabolic Integration:

  • Links between metabolic state and developmental progression through butyryl-CoA availability

  • Potential sensing mechanism where metabolic conditions influence developmental decisions via histone butyrylation

Studies demonstrate that the transition from spermatocytes to round spermatids involves significant changes in butyrylation patterns, suggesting developmental regulation of this modification in specialized cellular contexts .

What advanced mass spectrometry approaches can differentiate between closely related histone acylation marks including butyrylation?

Advanced mass spectrometry offers powerful approaches to distinguish histone acylations:

• High-Resolution LC-MS/MS Strategies:

  • Electron Transfer Dissociation (ETD): Preserves labile modifications and provides detailed fragmentation patterns

  • Parallel Reaction Monitoring (PRM): Enables precise targeting of specific modified peptides

  • SWATH-MS (Sequential Window Acquisition of all Theoretical Mass Spectra): Provides comprehensive data-independent acquisition for unbiased detection

• Chemical Derivatization Approaches:

  • Differential labeling strategies to distinguish butyrylation from acetylation

  • Selective chemical reactions targeting the extended carbon chain of butyryl groups

  • Isotopic labeling to track modification turnover rates

• Integrated Analytical Workflows:

  • Middle-down proteomics approaches analyzing larger histone fragments to preserve combinatorial modification patterns

  • Top-down proteomics for intact histone analysis to observe complete modification landscapes

  • Ion mobility separation to distinguish modifications with similar mass but different structures

• Quantitative Strategies:

  • SILAC (Stable Isotope Labeling with Amino acids in Cell culture) for precise relative quantification

  • Standard peptide-based absolute quantification approaches

  • Internal standard spike-in methods for cross-sample normalization

These advanced mass spectrometry approaches have been essential in demonstrating the existence and specificity of histone butyrylation as distinct from acetylation, enabling researchers to confidently distinguish these closely related modifications despite their structural similarities .

How does metabolism influence the dynamics of histone butyrylation compared to acetylation?

The metabolic regulation of histone butyrylation reveals important connections between cellular metabolism and epigenetic regulation:

• Metabolic Precursor Availability:

  • Butyrylation requires butyryl-CoA, derived primarily from fatty acid β-oxidation and specific amino acid catabolism

  • Acetylation uses acetyl-CoA, available from multiple metabolic pathways including glycolysis

  • The differential availability of these precursors under various metabolic conditions creates a mechanism for metabolism-responsive epigenetic regulation

• Enzyme Specificity and Competition:

  • Histone acetyltransferases (HATs) can sometimes utilize butyryl-CoA as an alternative substrate with varying efficiencies

  • Specialized acyltransferases may preferentially catalyze butyrylation

  • Deacylases (including HDACs and sirtuins) show different activities toward acetylated versus butyrylated substrates

• Nutritional Influences:

  • Dietary interventions affecting the butyryl-CoA/acetyl-CoA ratio can alter histone modification patterns

  • Fasting/feeding cycles produce distinct temporal patterns of acylation

  • Specialized diets (high-fat, ketogenic) may particularly affect butyrylation levels

• Metabolic Disease Connections:

  • Altered butyrylation patterns in metabolic disorders

  • Potential therapeutic interventions targeting butyrylation in metabolic diseases

  • Differential regulation in insulin-sensitive versus insulin-resistant states

This metabolic connection provides a mechanistic link between environmental factors (nutrition, activity) and gene regulation through differential histone modification patterns, with butyrylation potentially serving as a more specialized metabolic sensor than the more abundant acetylation .

What are the optimal sample preparation protocols for preserving Butyryl-HIST1H4A (K12) in different experimental contexts?

Preserving histone butyrylation requires specialized sample preparation approaches:

• Cell Culture Harvesting:

  • Avoid extended trypsinization (≤5 minutes) to prevent modification loss

  • Include deacylase inhibitors (10mM sodium butyrate, 10μM TSA) in all buffers

  • Process samples rapidly at cold temperatures (4°C)

  • Consider supplementing media with butyryl-CoA precursors prior to harvest

• Tissue Processing:

  • Flash-freeze tissues immediately after collection

  • Utilize specialized extraction buffers containing:

    • HDAC inhibitors (sodium butyrate, TSA, nicotinamide)

    • Protease inhibitor cocktail

    • Phosphatase inhibitors

    • Reducing agents to prevent oxidation

• Histone Extraction Methods:

  • Acid Extraction Protocol:

    • Lyse nuclei in 0.4N H₂SO₄ (15 minutes on ice)

    • Precipitate histones with TCA (33% final)

    • Wash with acetone containing 0.1% HCl followed by pure acetone

    • Maintain cold temperature throughout

  • High-Salt Extraction Alternative:

    • Use 420mM NaCl with HDAC inhibitors

    • Extract over 1 hour with gentle rotation

    • Filter through 0.45μm filter before downstream applications

• Preservation Strategies for Immunoprecipitation:

  • Use light cross-linking (0.1-0.3% formaldehyde, 5 minutes)

  • Include 10mM sodium butyrate in all ChIP buffers

  • Minimize washing steps and time

  • Consider specialized low-deacylation ChIP protocols

These optimized protocols have been demonstrated to effectively preserve butyrylation marks for subsequent analysis, with rapid processing and appropriate inhibitors being particularly critical for maintaining these potentially labile modifications .

How can sequential ChIP (re-ChIP) be optimized to study co-occurrence of butyrylation with other modifications?

Sequential ChIP optimization for butyrylation studies requires specialized techniques:

• Protocol Optimization for Butyrylation-Focused re-ChIP:

  • First IP Considerations:

    • Begin with the more abundant modification antibody

    • Use mild elution conditions to preserve butyrylation

    • Validate recovery efficiency with spike-in controls

  • Elution Methods:

    • DTT-based elution (10-20mM DTT, 30 minutes at 37°C)

    • Peptide competition elution for gentler release

    • Avoid harsh SDS elution when butyrylation is targeted

  • Second IP Adjustments:

    • Increase antibody concentration (1.5-2× standard amounts)

    • Extend incubation time (overnight at 4°C)

    • Add fresh deacylase inhibitors before second IP

• Cross-Validation Approaches:

  • Perform reciprocal re-ChIP (A→B and B→A)

  • Include single-IP controls alongside re-ChIP samples

  • Implement spike-in standards for quantitative assessment

• Analytical Considerations:

  • Account for cumulative efficiency loss in sequential steps

  • Develop statistical models for estimating co-occurrence

  • Use specialized normalization strategies for re-ChIP data

• Verification Strategies:

  • Mass spectrometry validation of re-ChIP material

  • Independent validation with proximity ligation assays

  • Complementary genetic approaches (histone mutants)

Studies using sequential ChIP have demonstrated that butyrylation at H4K5/K8 can occur alongside other modifications in specific genomic contexts, providing evidence for combinatorial histone modification patterns involving butyrylation .

What computational approaches can distinguish butyrylation-specific genomic patterns from acetylation patterns?

Advanced computational strategies for distinguishing butyrylation from acetylation patterns:

• Differential Peak Analysis Frameworks:

  • Implement specialized normalization strategies accounting for antibody efficiency differences

  • Use multivariate Hidden Markov Models to identify modification state transitions

  • Apply dynamic time warping algorithms for temporal pattern comparison

• Machine Learning Approaches:

  • Supervised classification algorithms to identify butyrylation-specific signatures

  • Unsupervised clustering to discover novel butyrylation-associated patterns

  • Deep learning models trained on validated datasets to predict butyrylation sites

• Integration of Multiple Data Types:

  • Multi-omics data integration frameworks combining:

    • ChIP-seq data for various modifications

    • RNA-seq transcriptional outputs

    • Metabolomic data reflecting precursor availability

    • Proteomic data on writer/eraser/reader proteins

• Feature Extraction and Pattern Recognition:

  • Positional analysis relative to genomic features

  • Sequence motif discovery around modification sites

  • Chromatin accessibility correlation analysis

  • Three-dimensional chromatin conformation integration

• Statistical Modeling of Modification Dynamics:

  • Bayesian approaches for modeling competing modifications

  • Time-series analysis for developmental transitions

  • Stochastic process modeling for modification state transitions

These computational approaches have revealed that butyrylation exhibits genomic distribution patterns distinct from acetylation, with enrichment at specific genomic elements and correlation with specialized gene expression programs rather than general transcriptional activation .