2-hydroxyisobutyryl-HIST1H3A (K56) Antibody

What is 2-hydroxyisobutyryl-HIST1H3A (K56) Antibody and what does it specifically detect?

This antibody specifically recognizes the 2-hydroxyisobutyrylation post-translational modification at lysine 56 (K56) on histone H3.1 (HIST1H3A). It is a polyclonal antibody raised in rabbits against a peptide sequence surrounding the 2-hydroxyisobutyryl-K56 site derived from human histone H3.1 . The antibody does not cross-react with unmodified H3K56 or other modifications at this site, making it valuable for studying this specific epigenetic mark. This modification is part of the expanding "histone code" that regulates chromatin structure and gene expression.

How does 2-hydroxyisobutyrylation at H3K56 differ from acetylation at the same site?

While both 2-hydroxyisobutyrylation and acetylation at H3K56 neutralize the positive charge of lysine, 2-hydroxyisobutyrylation introduces a bulkier chemical group that may have distinct functional consequences. Acetylation at H3K56 has been well-characterized in fungal species, where it blocks direct electrostatic interaction between histone H3 and nucleosomal DNA and is associated with sensitivity to genotoxic agents . 2-hydroxyisobutyrylation likely affects nucleosomal stability and DNA-histone interactions in a manner potentially distinct from acetylation, possibly recruiting different reader proteins. The availability of specific antibodies for each modification allows researchers to distinguish between these distinct epigenetic marks.

What are the validated applications for this antibody?

The 2-hydroxyisobutyryl-HIST1H3A (K56) antibody has been validated for multiple research applications, including:

• Western Blot (WB): Validated at dilutions of 1:100-1:1000

• Enzyme-Linked Immunosorbent Assay (ELISA): Validated for detection

• Immunofluorescence (IF): Recommended dilutions of 1:10-1:100

• Immunocytochemistry (ICC): Validated at dilutions of 1:20-1:200

• Chromatin Immunoprecipitation (ChIP): Validated for use

When selecting application-specific dilutions, researchers should first perform optimization tests using positive control samples known to contain the 2-hydroxyisobutyryl-K56 modification.

What is the recommended protocol for using this antibody in Western blot experiments?

For optimal Western blot results with 2-hydroxyisobutyryl-HIST1H3A (K56) antibody:

• Sample preparation: Extract histones using acid extraction methods to enrich for histone proteins. For cell samples, use approximately 1-5×10^6 cells per lane.

• Gel electrophoresis: Use 15-18% SDS-PAGE gels to properly resolve histone proteins (typically 15-20 kDa).

• Transfer: Employ PVDF membranes (0.2 μm pore size) and transfer at 30V overnight at 4°C for optimal histone transfer.

• Blocking: Block with 5% non-fat dry milk or BSA in TBST for 1 hour at room temperature.

• Primary antibody incubation: Dilute the antibody 1:100-1:1000 in blocking buffer and incubate overnight at 4°C .

• Detection: Use appropriate HRP-conjugated secondary antibodies and ECL detection systems.

• Controls: Include both positive controls (samples known to contain the modification) and negative controls (samples treated with demethylase enzymes).

Expected results should show a band at approximately 15-17 kDa corresponding to histone H3.1.

What are the critical considerations for immunofluorescence experiments with this antibody?

For successful immunofluorescence using 2-hydroxyisobutyryl-HIST1H3A (K56) antibody:

• Fixation: Use 4% paraformaldehyde for 15 minutes followed by permeabilization with 0.2% Triton X-100 for 10 minutes.

• Blocking: Block with 3-5% BSA or normal serum in PBS for 1 hour at room temperature.

• Primary antibody: Dilute the antibody 1:10-1:100 in blocking buffer and incubate overnight at 4°C .

• Secondary antibody: Use fluorophore-conjugated anti-rabbit secondary antibodies at appropriate dilutions (typically 1:200-1:1000).

• Nuclear counterstaining: DAPI (1 μg/mL) is recommended for nuclear visualization.

• Controls: Include secondary-only controls and competitive blocking with the immunizing peptide when possible.

• Image acquisition: Use confocal microscopy for optimal visualization of nuclear staining patterns.

Expected nuclear staining patterns may vary depending on cell type and physiological state, with potentially enriched signals during S-phase based on knowledge of H3K56 acetylation dynamics .

How should this antibody be validated for specificity in experimental systems?

To validate antibody specificity:

• Peptide competition assays: Pre-incubate the antibody with increasing concentrations of the immunizing peptide (containing 2-hydroxyisobutyryl-K56) prior to application in your experiment. Signal reduction confirms specificity.

• Comparison with other modification-specific antibodies: Compare staining patterns with antibodies against unmodified H3K56 or H3K56ac to identify unique patterns.

• Knockdown/knockout validation: Use cells/tissues with reduced expression of enzymes responsible for 2-hydroxyisobutyrylation as negative controls.

• Mass spectrometry correlation: Validate findings with orthogonal techniques like mass spectrometry to confirm the presence of 2-hydroxyisobutyryl-K56.

• Testing against recombinant proteins: Use recombinant histones with and without the modification as controls.

This multi-faceted approach ensures that observed signals truly represent the targeted modification.

How does histone chaperone activity influence H3K56 modifications and antibody detection?

Histone chaperones play crucial roles in facilitating histone modifications. For H3K56 acetylation, the histone chaperone Asf1 is essential, forming a complex with the acetyltransferase Rtt109 . This complex formation is necessary because:

• Asf1 helps unwind the histone H3 α-N terminal region where K56 is located, making it accessible to modifying enzymes .

• The chaperone stabilizes the C-terminal β-strand of histone H4, which is a prerequisite for H3K56 modification .

• The multiprotein complex enables multisite substrate recognition, essential for specific targeting of H3K56 .

When using the 2-hydroxyisobutyryl-HIST1H3A (K56) antibody, researchers should consider that chaperone activity may affect modification levels and thus antibody detection. Cellular stress, cell cycle stage, or experimental manipulations that alter chaperone function could impact detected signal intensity independent of the actual enzymatic activity responsible for 2-hydroxyisobutyrylation.

What is the current understanding of the biological significance of H3K56 2-hydroxyisobutyrylation versus acetylation?

While H3K56 acetylation is well-characterized and known to be critical for DNA replication-coupled nucleosome assembly and genome stability , the specific biological significance of 2-hydroxyisobutyrylation at this same site is still emerging. Based on the current understanding of histone modifications:

• Structural implications: Both modifications neutralize the positive charge of lysine, but 2-hydroxyisobutyrylation introduces a bulkier group that may create distinct changes in chromatin structure.

• Reader protein recruitment: Different modifications likely recruit different reader proteins, activating distinct downstream pathways.

• Cell cycle regulation: Like acetylation, 2-hydroxyisobutyrylation may show cell cycle-dependent patterns, particularly during DNA replication when H3K56 is most accessible.

• DNA damage response: H3K56ac is associated with DNA damage response ; 2-hydroxyisobutyrylation may have similar or complementary roles.

Researchers using this antibody can contribute to this understanding by characterizing the dynamics of this modification across different cellular contexts and in response to various stimuli.

How can I design experiments to investigate the relationship between H3K56 2-hydroxyisobutyrylation and gene expression?

To investigate relationships between H3K56 2-hydroxyisobutyrylation and gene expression:

• ChIP-seq approach:

  • Perform ChIP-seq using the 2-hydroxyisobutyryl-HIST1H3A (K56) antibody

  • Correlate modification distribution with gene expression data from RNA-seq

  • Analyze enrichment at specific genomic features (promoters, enhancers, gene bodies)

• Perturbation studies:

  • Identify and modulate enzymes responsible for deposition/removal of this modification

  • Monitor changes in gene expression following perturbation

  • Perform rescue experiments to confirm specificity

• Time-course experiments:

  • Track modification dynamics during biological processes (cell cycle, differentiation)

  • Correlate temporal changes with gene expression alterations

• Comparison with other modifications:

  • Perform sequential ChIP (re-ChIP) to identify co-occurrence with other modifications

  • Create modification interaction maps to understand the broader epigenetic context

These approaches can help establish whether H3K56 2-hydroxyisobutyrylation has activating or repressive effects on transcription, and in which genomic contexts.

What are the optimal storage and handling conditions for maintaining antibody performance?

To maintain optimal performance of the 2-hydroxyisobutyryl-HIST1H3A (K56) antibody:

• Storage temperature: Store at -20°C or -80°C for long-term storage .

• Buffer composition: The antibody is typically supplied in a buffer containing 50% glycerol, 0.01 M PBS, pH 7.4, with 0.03% Proclin 300 as a preservative .

• Aliquoting: Upon receipt, prepare small working aliquots to avoid repeated freeze-thaw cycles which can damage antibody performance.

• Thawing procedure: Thaw on ice and centrifuge briefly before use to ensure homogeneity.

• Working dilution preparation: Prepare working dilutions fresh on the day of experiment for optimal results.

• Shipping conditions: The antibody is typically shipped on blue ice ; verify condition upon arrival.

• Expiration: Follow manufacturer recommendations for expiration dates, typically 12-24 months from date of receipt when properly stored.

Adhering to these conditions will help ensure consistent and reliable experimental results.

What are common troubleshooting issues when using this antibody and how can they be resolved?

For persistent issues, peptide competition assays can help determine if signals are specific to the 2-hydroxyisobutyryl-K56 modification.

How can I quantitatively analyze Western blot or immunofluorescence data with this antibody?

For quantitative analysis:

• Western blot quantification:

  • Always include loading controls (total H3 or another stable protein)

  • Use signal normalization: (2-hydroxyisobutyryl-K56 signal)/(total H3 signal)

  • Employ linear detection methods (fluorescent secondary antibodies rather than ECL when possible)

  • Ensure exposure times are within the linear range of detection

  • Use technical and biological replicates (minimum n=3)

• Immunofluorescence quantification:

  • Measure nuclear fluorescence intensity using appropriate imaging software

  • Analyze minimum 50-100 cells per condition

  • Control for background by subtracting non-specific signal

  • Normalize to nuclear area or DNA content (DAPI signal)

  • Report distribution of signals (not just means) as modification levels may vary across cell populations

• Statistical analysis:

  • Use appropriate statistical tests based on data distribution

  • For multiple comparisons, apply corrections (e.g., Bonferroni, FDR)

  • Report effect sizes alongside p-values

These approaches ensure rigorous and reproducible quantification of 2-hydroxyisobutyryl-K56 levels across experimental conditions.

How does 2-hydroxyisobutyryl-HIST1H3A (K56) relate to other histone modifications in the broader epigenetic landscape?

Understanding the relationship between 2-hydroxyisobutyryl-K56 and other histone modifications requires contextualizing this mark within the broader histone code:

• Modification crosstalk: Evidence from studies on H3K56 acetylation suggests potential crosstalk with other modifications. H3K56 resides at the entry-exit points of DNA in the nucleosome, making it structurally significant for DNA-histone interactions .

• Temporal dynamics: Like H3K56 acetylation, which shows cell cycle-dependent patterns (particularly during S-phase in fungi) , 2-hydroxyisobutyrylation may have specific temporal dynamics that coordinate with other modifications.

• Functional complementarity: The presence of multiple possible modifications at K56 (acetylation, 2-hydroxyisobutyrylation, potentially others) suggests functional specialization or contextual regulation.

• Species-specific patterns: While H3K56 acetylation is well-characterized in fungal species , the prevalence and significance of 2-hydroxyisobutyrylation may vary across species and cell types.

Researchers should consider investigating the co-occurrence or mutual exclusivity of 2-hydroxyisobutyryl-K56 with other histone marks using sequential ChIP or mass spectrometry approaches.

What technologies beyond conventional antibody-based methods can be used to study H3K56 2-hydroxyisobutyrylation?

Beyond antibody-based detection, several advanced technologies can enhance the study of H3K56 2-hydroxyisobutyrylation:

• Mass spectrometry:

  • Targeted MS approaches can quantify 2-hydroxyisobutyryl-K56 with high precision

  • Bottom-up proteomics for modification identification

  • Top-down approaches to analyze combinatorial patterns with other modifications

• Synthetic biology approaches:

  • Designer histones with site-specific incorporation of 2-hydroxyisobutyryl-lysine

  • CRISPR-based epigenome editing to manipulate this modification

• Structural biology:

  • Cryo-EM to visualize nucleosome structure with this modification

  • Hydrogen-deuterium exchange mass spectrometry to assess structural impacts

• Proximity labeling:

  • BioID or APEX2 fusions to identify proteins that recognize this modification

• Single-molecule approaches:

  • FRET-based assays to monitor dynamic changes in chromatin structure upon modification

These complementary approaches, when combined with antibody-based methods, provide a more comprehensive understanding of the functional significance of this modification.

How can I investigate the enzymes responsible for deposition and removal of H3K56 2-hydroxyisobutyrylation?

To identify and characterize the enzymatic machinery governing H3K56 2-hydroxyisobutyrylation:

• Candidate approach:

  • Test known histone-modifying enzymes through overexpression/knockdown

  • Begin with enzymes known to modify H3K56 with other marks (e.g., Rtt109 for acetylation)

  • Examine enzymes known to catalyze 2-hydroxyisobutyrylation at other sites

• Unbiased screens:

  • CRISPR screens with 2-hydroxyisobutyryl-K56 levels as readout

  • Chemical inhibitor libraries to identify pathways regulating this modification

  • Proteomic approaches to identify proteins that interact with modified H3K56

• Biochemical characterization:

  • In vitro reconstitution of enzymatic activity

  • Structural studies of enzyme-substrate complexes

  • Kinetic analyses to determine enzyme specificities

• Metabolic connections:

  • Investigate links to cellular metabolism, particularly pathways producing 2-hydroxyisobutyryl-CoA

  • Isotope tracing to track metabolic precursors to histone modification

Understanding the enzymatic regulation will provide insights into the physiological contexts where this modification is relevant and potential therapeutic targets in diseases where epigenetic dysregulation occurs.