Butyrly-HIST1H4A (K8) Antibody

What is the Butyryl-HIST1H4A (K8) antibody and what specific modification does it recognize?

The Butyryl-HIST1H4A (K8) antibody is a specialized immunoglobulin that specifically recognizes histone H4 proteins containing butyrylation at the lysine 8 position (K8). This post-translational modification is part of the histone code that regulates chromatin structure and gene expression. The antibody binds to the peptide sequence surrounding the butyrylated K8 residue of human histone H4, allowing researchers to detect this specific epigenetic mark in various experimental contexts . Histone butyrylation is distinct from acetylation and represents an important regulatory mechanism in chromatin biology, with the K8 position being particularly significant for transcriptional regulation .

What validated applications are available for Butyryl-HIST1H4A (K8) antibody?

The Butyryl-HIST1H4A (K8) antibody has been validated for multiple experimental applications, providing researchers with versatile options for studying this histone modification:

These applications have been verified through rigorous validation procedures, ensuring specificity for the butyrylated K8 epitope across different experimental contexts .

How should researchers properly store and handle the Butyryl-HIST1H4A (K8) antibody to maintain its activity?

Proper storage and handling of the Butyryl-HIST1H4A (K8) antibody are crucial for maintaining its specificity and activity. The antibody is typically supplied in liquid form containing 50% glycerol and 0.01M PBS (pH 7.4) with 0.03% Proclin 300 as a preservative . For optimal stability, researchers should adhere to the following guidelines:

• Upon receipt, aliquot the antibody in small volumes (10-20 μl) to minimize freeze-thaw cycles.

• Store at -20°C or preferably -80°C for long-term preservation.

• Avoid repeated freeze-thaw cycles as this can significantly reduce antibody activity and specificity.

• When using, thaw aliquots on ice and keep cold during experimental procedures.

• Return unused portions to -20°C or -80°C immediately after use.

• For diluted working solutions, prepare fresh on the day of use or store at 4°C for no more than 24 hours.

Following these storage recommendations will help ensure consistent antibody performance across experiments and extend the usable life of the reagent .

What is the species reactivity profile of the Butyryl-HIST1H4A (K8) antibody?

The Butyryl-HIST1H4A (K8) antibody demonstrates a defined species reactivity profile that researchers should consider when planning experiments:

What controls should be included when using Butyryl-HIST1H4A (K8) antibody for experimental validation?

When designing experiments with the Butyryl-HIST1H4A (K8) antibody, incorporating appropriate controls is essential for result validation and troubleshooting:

• Positive Control: Include samples known to contain butyrylated H4K8, such as histone extracts from cells treated with butyrate or butyrylation-promoting compounds.

• Negative Control:

  • Peptide competition assay: Pre-incubate the antibody with the butyrylated peptide immunogen to block specific binding.

  • Use histone extracts from cells where H4K8 butyrylation has been depleted (e.g., through HDAC/KDAC treatment).

• Specificity Controls:

  • Test reactivity against unmodified H4K8 peptides.

  • Test reactivity against H4K8 with other modifications (acetylation, methylation).

  • Use lysine-to-alanine substitution mutants (K8A) in overexpression systems .

• Antibody Controls:

  • Include an isotype control (rabbit IgG) at the same concentration.

  • Perform the experiment without primary antibody.

• Loading/Normalization Controls:

  • For Western blots, include antibodies against total histone H4 or other housekeeping proteins.

  • For ChIP experiments, include input samples and IgG controls.

These controls help confirm the specificity of the antibody for butyrylated H4K8 and validate experimental findings .

How can researchers distinguish between butyrylation and other acylation modifications at H4K8 using antibody-based methods?

Distinguishing between butyrylation and other acylation modifications (such as acetylation, propionylation, or crotonylation) at H4K8 requires careful experimental design:

• Antibody Specificity Validation:

  • Perform peptide competition assays using synthetic peptides containing specific modifications.

  • Conduct ELISA tests with modified peptide arrays containing H4K8 with various acylations.

  • Compare reactivity patterns of the Butyryl-HIST1H4A (K8) antibody with antibodies specific for other modifications.

• Mass Spectrometry Correlation:

  • Validate antibody-based findings with mass spectrometry analysis to confirm the precise modification.

  • Examine the mass shift associated with butyrylation (70 Da) versus acetylation (42 Da) or propionylation (56 Da).

• Enzymatic Manipulation:

  • Treatment with modification-specific eraser enzymes (HDACs/KDACs with substrate preferences).

  • Manipulation of cellular pathways that specifically affect butyrylation (e.g., butyrate treatment or butyryl-CoA metabolism).

• Immunofluorescence Colocalization:

  • Perform dual labeling with antibodies against different modifications to analyze colocalization patterns.

Some antibodies show cross-reactivity between closely related modifications, particularly between butyrylation and acetylation. The literature indicates that high-quality Butyryl-HIST1H4A (K8) antibodies should show less than 5% cross-reactivity with the acetylated form, but this should be verified experimentally for each specific application .

What are the optimal conditions for using Butyryl-HIST1H4A (K8) antibody in ChIP experiments?

Optimizing ChIP experiments with Butyryl-HIST1H4A (K8) antibody requires careful attention to several parameters:

• Crosslinking Conditions:

  • Use 1% formaldehyde for 10 minutes at room temperature for standard crosslinking.

  • For detection of transient or weak interactions, consider using dual crosslinkers (formaldehyde plus disuccinimidyl glutarate).

• Chromatin Fragmentation:

  • Target fragment sizes of 200-500 bp for optimal resolution.

  • For sonication: 10-15 cycles (30 seconds ON/30 seconds OFF) using a Bioruptor or similar device.

  • For enzymatic digestion: Optimize MNase concentration and digestion time for consistent fragmentation.

• Antibody Amounts and Incubation:

  • Use 2-5 μg of Butyryl-HIST1H4A (K8) antibody per ChIP reaction.

  • Incubate overnight at 4°C with rotation for optimal binding.

  • Pre-clear chromatin with protein A/G beads to reduce background.

• Washing Conditions:

  • Use increasingly stringent wash buffers to reduce non-specific binding.

  • Typically: Low salt wash → High salt wash → LiCl wash → TE wash.

• Elution and Reversal of Crosslinks:

  • Elute with 1% SDS, 0.1M NaHCO3 at 65°C.

  • Reverse crosslinks at 65°C for 4-6 hours or overnight.

The Butyryl-HIST1H4A (K8) antibody has been validated for ChIP applications across multiple suppliers, making it suitable for studying genomic distribution of this modification . Research has shown that butyrylation marks, like acetylation, are often enriched at transcription start sites, suggesting roles in gene activation .

How does cell cycle progression affect H4K8 butyrylation levels and what implications does this have for experimental design?

H4K8 butyrylation exhibits dynamic changes during the cell cycle, which has important implications for experimental design:

• Cell Cycle-Dependent Patterns:

  • Similar to acetylation marks, H4K8 butyrylation levels typically decrease during mitosis when chromatin is highly condensed.

  • Levels increase during G1 and S phases, correlating with chromatin decondensation and active transcription.

  • Newly synthesized histones incorporated during S phase often carry specific modification patterns, including butyrylation marks.

• Experimental Design Considerations:

  • Cell Synchronization: When comparing samples, synchronize cells to the same cell cycle phase using methods like double thymidine block, nocodazole treatment, or serum starvation/release.

  • Flow Cytometry Correlation: Consider combining immunostaining for H4K8bu with DNA content analysis to correlate modifications with cell cycle phases.

  • Time Course Studies: Design experiments to track butyrylation changes across the cell cycle, sampling at defined intervals.

• Interpretation Challenges:

  • Changes in observed butyrylation levels might reflect cell cycle distribution differences rather than treatment effects.

  • In asynchronous populations, results represent an average across all cell cycle phases.

Research on histone H4 modifications indicates that some acetylation patterns on newly assembled histones differ from those on histones in mature chromatin, with specific combinations (like K5/K12 diacetylation) being markers of new histone incorporation. Similar patterns may exist for butyrylation marks, suggesting researchers should consider histone deposition timing when interpreting results .

What is the relationship between H4K8 butyrylation and H4K8 acetylation, and how can researchers differentiate their functional impacts?

H4K8 butyrylation and acetylation represent related but distinct modifications with potentially different functional impacts:

• Structural and Chemical Differences:

  • Butyrylation adds a four-carbon chain (butyrate group), while acetylation adds a two-carbon chain (acetate group).

  • The longer carbon chain of butyrylation creates a more hydrophobic modification that may influence protein-protein interactions differently.

• Distribution Patterns:

  • Both modifications can be found at active gene promoters and enhancers.

  • ChIP-seq studies indicate that H4K8 acetylation is enriched around transcription start sites, and preliminary evidence suggests butyrylation may show similar enrichment patterns .

  • The relative abundance typically favors acetylation over butyrylation in most cellular contexts.

• Differentiation Strategies:

  • Sequential ChIP (ReChIP): Perform ChIP with anti-H4K8ac followed by ChIP with anti-H4K8bu (or vice versa) to identify regions with co-occurrence or exclusivity.

  • Manipulate Cellular Metabolism: Altering levels of acetyl-CoA versus butyryl-CoA can shift the balance between modifications.

  • Enzyme Specificity: Identify and manipulate enzymes with preferential activity for butyrylation versus acetylation.

  • Temporal Analysis: Track modifications during dynamic processes like cell differentiation or response to stimuli.

• Functional Readout Assays:

  • Reporter gene assays with histone mutants (K8R or K8Q) to prevent modification.

  • Identification of specific reader proteins that preferentially bind butyrylated versus acetylated H4K8.

Understanding the relationship between these modifications is an active research area, with evidence suggesting they may work cooperatively in some contexts but have distinct functions in others. The differential recognition by reader proteins may be particularly important for determining their specific roles in transcriptional regulation .

What are the best practices for optimizing Western blot protocols when using Butyryl-HIST1H4A (K8) antibody?

Optimizing Western blot protocols for detecting H4K8 butyrylation requires specific adjustments to standard procedures:

• Sample Preparation:

  • Extract histones using specialized acid extraction methods (e.g., 0.2N HCl extraction or commercial histone extraction kits).

  • Include histone deacetylase inhibitors (e.g., sodium butyrate, trichostatin A) and protease inhibitors in lysis buffers.

  • Consider using histone-enriched fractions rather than whole cell lysates for better signal-to-noise ratio.

• Gel Electrophoresis:

  • Use high-percentage (15-18%) SDS-PAGE gels or specialized Triton-Acid-Urea (TAU) gels to properly resolve the low molecular weight histone proteins.

  • Load 2-5 μg of purified histones or 15-20 μg of acid-extracted histones.

• Transfer Conditions:

  • Optimize transfer of small proteins using PVDF membranes (0.2 μm pore size).

  • Consider semi-dry transfer systems with specialized buffers for small proteins.

  • Transfer at lower voltage (25V) for longer times (2 hours) to prevent proteins from passing through the membrane.

• Blocking and Antibody Incubation:

  • Block with 5% BSA rather than milk (milk contains proteins that can bind to some histone antibodies).

  • Use optimized dilution of Butyryl-HIST1H4A (K8) antibody (typically 1:500 to 1:2000).

  • Extend primary antibody incubation to overnight at 4°C for better sensitivity.

• Signal Detection Optimization:

  • Use high-sensitivity ECL substrates or fluorescent secondary antibodies.

  • Consider signal enhancement systems for low-abundance modifications.

• Controls and Normalization:

  • Strip and reprobe with antibodies against total H4 or H3 for normalization.

  • Include positive controls (butyrate-treated cells) and negative controls (deacetylase-treated extracts).

These optimizations help overcome common challenges in detecting histone modifications by Western blot, including the small size of histones (H4 is approximately 11 kDa), potential cross-reactivity with other modifications, and relatively low abundance of specific butyrylation marks .

How can researchers address potential cross-reactivity between the Butyryl-HIST1H4A (K8) antibody and other histone modifications in ChIP-seq experiments?

Addressing cross-reactivity concerns in ChIP-seq experiments requires multi-faceted validation and analytical approaches:

• Comprehensive Antibody Validation:

  • Perform peptide array testing against a panel of modified histone peptides, particularly those with similar modifications at K8 and nearby residues.

  • Conduct sequential ChIP experiments using antibodies against potentially cross-reactive modifications to determine overlap.

  • Include spike-in controls with synthetic modified nucleosomes containing defined modifications.

• Bioinformatic Analysis Strategies:

  • Compare ChIP-seq profiles with datasets for similar modifications (H4K8ac, H4K5bu, H4K12bu).

  • Apply differential binding analysis to identify regions uniquely enriched for H4K8bu.

  • Utilize machine learning algorithms trained on validated modification sites to identify characteristic features of true H4K8bu peaks.

• Experimental Validation of ChIP-seq Findings:

  • Perform targeted ChIP-qPCR on selected genomic regions with primer sets spanning peak regions.

  • Validate with orthogonal methods such as CUT&RUN or CUT&Tag which offer higher resolution.

  • Correlate genome-wide distribution with mass spectrometry-based mapping of modifications.

• Controls for Data Interpretation:

  • Include IgG controls and input normalization in all analyses.

  • Perform ChIP-seq in systems with modulated levels of H4K8 butyrylation (e.g., HDAC inhibitor treatment, butyryl-CoA modulating conditions).

  • Compare binding profiles in wild-type versus K8R mutant histone backgrounds when possible.

Research indicates that even highly specific antibodies may exhibit some degree of cross-reactivity under ChIP conditions where the chromatin environment can affect epitope accessibility. Rigorous validation is therefore essential for confident interpretation of genome-wide butyrylation patterns .

What are the methodological considerations for investigating the relationship between H4K8 butyrylation and transcriptional regulation?

Investigating the relationship between H4K8 butyrylation and transcriptional regulation requires sophisticated methodological approaches:

• Integrative Genomic Analysis:

  • Combine H4K8bu ChIP-seq with RNA-seq to correlate modification patterns with transcriptional outputs.

  • Perform time-course experiments to establish temporal relationships between butyrylation changes and transcriptional responses.

  • Integrate with data on transcription factor binding, chromatin accessibility (ATAC-seq), and other histone modifications.

• Mechanistic Investigation:

  • Identify and characterize reader proteins that specifically recognize H4K8bu using techniques such as:

    • Peptide pull-downs coupled with mass spectrometry

    • CRISPR screens for factors affecting H4K8bu-dependent gene expression

    • Proximity labeling methods (BioID, APEX) to identify proteins associated with H4K8bu-marked chromatin

• Causal Relationship Testing:

  • Engineer systems for targeted manipulation of H4K8 butyrylation at specific loci:

    • dCas9-fusion proteins with butyryl-transferase activity

    • Optogenetic or chemical-inducible systems for temporally controlled modification

  • Analyze transcriptional consequences of site-specific butyrylation manipulation using reporter assays or endogenous gene expression analysis.

• Context-Dependent Functionality:

  • Examine H4K8bu distribution and function across different:

    • Cell types and differentiation states

    • Metabolic conditions affecting butyryl-CoA availability

    • Stress responses and environmental stimuli

• Single-Cell Approaches:

  • Utilize single-cell technologies to address heterogeneity in butyrylation patterns:

    • scCUT&Tag for single-cell profiling of modifications

    • Correlation with scRNA-seq to link modification patterns to transcriptional states at the single-cell level

Current research suggests that, like acetylation, H4K8 butyrylation is associated with transcriptionally active regions, particularly around transcription start sites, but may have distinct functions in specific genomic contexts or cellular states . Understanding these context-specific functions requires multidimensional experimental approaches combining genomic, biochemical, and genetic methods.

How can researchers effectively use Butyryl-HIST1H4A (K8) antibody to study the interplay between butyrylation and other histone modifications?

Studying the interplay between H4K8 butyrylation and other histone modifications requires specialized approaches that can capture modification co-occurrence and functional relationships:

• Multi-modification Analysis Techniques:

  • Sequential ChIP (ReChIP): Perform initial ChIP with Butyryl-HIST1H4A (K8) antibody followed by a second round with antibodies against other modifications to identify co-occurrence.

  • Mass Spectrometry of Histone PTMs: Use middle-down or top-down MS approaches to identify combinatorial patterns of modifications on the same histone tail.

  • Multiplexed Imaging: Apply multi-color immunofluorescence with antibodies against different modifications to analyze spatial relationships.

• Perturbation Strategies:

  • Systematically inhibit or activate enzymes responsible for specific modifications:

    • HDAC inhibitors (TSA, SAHA, butyrate)

    • HAT inhibitors (C646, A-485)

    • Methyltransferase inhibitors (chaetocin, UNC1999)

  • Monitor how altering one modification affects H4K8 butyrylation patterns genome-wide.

• Nucleosome-Resolution Analysis:

  • Use MNase-ChIP to analyze modification patterns at nucleosome resolution.

  • Apply paired-end sequencing to determine the precise positioning of modified nucleosomes.

  • Analyze modification asymmetry between nucleosomes (e.g., H3K4me3 on one nucleosome, H4K8bu on adjacent nucleosome).

• Computational Integration:

  • Develop machine learning approaches to identify patterns of modification co-occurrence.

  • Apply network analysis to model relationships between different modifications.

  • Use predictive modeling to generate hypotheses about modification interdependencies.

• Physical Interaction Analysis:

  • Investigate interactions between enzymes that deposit or remove H4K8bu and other chromatin-modifying complexes.

  • Study competition or cooperation between readers of different modifications.

Research suggests that butyrylation may function within histone modification networks, potentially acting in concert with or antagonistically to other modifications. For example, butyrylation might compete with acetylation for the same lysine residues or work synergistically with other activation-associated marks like H3K4me3 . Understanding these relationships is crucial for deciphering the histone code and its role in transcriptional regulation.

What strategies can researchers employ to investigate the enzymes responsible for regulating H4K8 butyrylation dynamics using the Butyryl-HIST1H4A (K8) antibody?

Investigating enzymes that regulate H4K8 butyrylation requires systematic approaches combining biochemical, genetic, and genomic methods:

• Writer Enzyme Identification and Characterization:

  • Candidate Approach: Test known histone acetyltransferases (HATs) for butyrylation activity using in vitro assays with recombinant enzymes and detecting products with Butyryl-HIST1H4A (K8) antibody.

  • Unbiased Screening: Perform CRISPR screens targeting chromatin-modifying enzymes, measuring global H4K8bu levels by immunoblotting or ChIP-seq.

  • Metabolic Manipulation: Alter cellular butyryl-CoA levels through media supplementation or metabolic pathway perturbation, then assess effects on H4K8bu.

• Eraser Enzyme Analysis:

  • HDAC Inhibitor Studies: Systematically test the effects of specific HDAC/KDAC inhibitors on H4K8bu levels.

  • Recombinant Enzyme Assays: Test deacylase activity of purified HDACs against H4K8bu substrates, detecting remaining modification with the antibody.

  • Enzyme Knockdown/Knockout: Create genetic models with reduced expression of candidate erasers, then perform ChIP-seq to map changes in H4K8bu distribution.

• Enzyme-Substrate Interaction Analysis:

  • Chromatin Association Studies: Perform ChIP-seq for candidate enzymes and correlate their genomic localization with H4K8bu patterns.

  • Proximity Labeling: Use BioID or APEX2 fusions with candidate enzymes to identify chromatin regions in close proximity.

  • Domain Mutation Analysis: Create enzyme variants with mutations in catalytic or substrate recognition domains and assess their impact on H4K8bu.

• Dynamic Regulation Studies:

  • Stimulus Response: Apply cellular stresses or signaling activators and track temporal changes in H4K8bu using ChIP-seq or immunofluorescence time courses.

  • Development and Differentiation: Profile H4K8bu during cellular differentiation to identify stage-specific changes in modification patterns.

  • Environmental Influence: Investigate how nutrient availability (particularly short-chain fatty acids) affects enzyme activity and H4K8bu levels.

• Enzyme-Specific Inhibitor Development:

  • Use the Butyryl-HIST1H4A (K8) antibody to develop high-throughput screening assays for compounds that specifically modulate H4K8 butyrylation.

  • Validate candidate inhibitors with in vitro enzymatic assays and cellular H4K8bu profiling.

Recent research has implicated several histone acetyltransferases (including p300/CBP) as potential butyryl-transferases, and certain HDACs (particularly SIRT1, SIRT2, and HDAC3) as debutyrylases, but the complete enzymatic landscape remains to be fully characterized . The Butyryl-HIST1H4A (K8) antibody provides a critical tool for these investigations by enabling specific detection of the modification in various experimental contexts.

How can researchers effectively validate and troubleshoot unexpected Butyryl-HIST1H4A (K8) antibody staining patterns in immunofluorescence experiments?

Validating and troubleshooting unexpected staining patterns with Butyryl-HIST1H4A (K8) antibody requires systematic investigation:

• Pattern Characterization and Documentation:

  • Thoroughly document the unexpected pattern (nuclear vs. cytoplasmic, focal vs. diffuse, cell cycle dependency).

  • Quantify the frequency of the pattern across different cell types and conditions.

  • Compare to published patterns for other histone H4 modifications.

• Technical Validation:

  • Fixation Method Comparison: Test multiple fixation protocols (paraformaldehyde, methanol, acetone) as fixation can affect epitope accessibility.

  • Antigen Retrieval Optimization: Evaluate different antigen retrieval methods (heat-induced, enzymatic, pH variations).

  • Blocking Conditions: Test alternative blocking agents (BSA, normal serum, commercial blockers) to reduce non-specific binding.

  • Antibody Titration: Perform a dilution series (1:50 to 1:1000) to identify optimal signal-to-noise ratio.

  • Detection System Comparison: Compare different secondary antibodies and visualization methods (direct vs. amplified).

• Specificity Confirmation:

  • Peptide Competition: Pre-incubate antibody with butyrylated and non-butyrylated H4K8 peptides to demonstrate signal specificity.

  • Multiple Antibody Validation: Test alternative anti-H4K8bu antibodies from different sources/clones.

  • Genetic Controls: Use H4K8R mutant-expressing cells or CRISPR-engineered K8 mutant cells as negative controls.

  • Enzyme Manipulation: Treat cells with deacetylase inhibitors or butyryl-CoA modulators to alter modification levels.

• Biological Context Investigation:

  • Cell Cycle Analysis: Co-stain with cell cycle markers to determine if the pattern correlates with specific cell cycle phases.

  • Stress Response: Evaluate if the pattern changes under different cellular stresses (oxidative, genotoxic, metabolic).

  • Co-localization Studies: Perform dual staining with markers for nuclear compartments (nucleoli, PML bodies, transcription factories).

  • Super-resolution Microscopy: Apply techniques like STORM or STED to resolve fine details of the staining pattern.

• Alternative Approach Correlation:

  • Validate findings with orthogonal techniques (ChIP-seq, mass spectrometry).

  • Consider that unexpected patterns might represent novel biological insights rather than technical artifacts.

Unexpected staining patterns sometimes reflect genuine biological phenomena rather than technical issues. For example, some histone modifications show dramatic redistribution during cellular processes like mitosis, DNA damage response, or cellular senescence . Careful validation allows researchers to distinguish between technical artifacts and potentially important biological discoveries.