2-hydroxyisobutyryl-HIST1H2BC (K57) Antibody

What is the 2-hydroxyisobutyryl-HIST1H2BC (K57) Antibody and what does it target?

The 2-hydroxyisobutyryl-HIST1H2BC (K57) Antibody is a rabbit polyclonal antibody specifically designed to detect the 2-hydroxyisobutyrylation modification at lysine 57 of the human Histone H2B type 1-C/E/F/G/I protein. This antibody recognizes a post-translational modification that is part of the expanding "histone code" involved in chromatin regulation. The antibody was developed using a synthesized peptide derived from Human Histone H2B type 1-C/E/F/G/I protein (amino acids 53-64) . This specific modification is emerging as an important epigenetic marker that may regulate gene expression through altering chromatin structure and DNA accessibility.

How does 2-hydroxyisobutyrylation differ from other histone modifications like butyrylation?

2-hydroxyisobutyrylation differs structurally and functionally from other acylation modifications such as butyrylation. While butyrylation (like the H2B butyryl K5 modification) involves the addition of a butyryl group (CH₃CH₂CH₂CO-) to lysine residues , 2-hydroxyisobutyrylation involves the addition of a 2-hydroxyisobutyryl group ((CH₃)₂C(OH)CO-) to lysine residues. This structural difference results in distinct biological functions, protein interactions, and enzymatic regulation. The 2-hydroxyisobutyryl modification creates a unique binding surface on the histone that recruits specific reader proteins, potentially leading to different downstream effects on chromatin structure and gene expression compared to butyrylation or other modifications.

What experimental applications has the 2-hydroxyisobutyryl-HIST1H2BC (K57) Antibody been validated for?

The 2-hydroxyisobutyryl-HIST1H2BC (K57) Antibody has been validated for several key research applications:

• Enzyme-Linked Immunosorbent Assay (ELISA)

• Immunofluorescence (IF)

• Immunocytochemistry (ICC)

While these are the specifically tested applications, researchers might also explore its utility in other immunological techniques based on the validation of similar histone modification antibodies, such as Western blotting and Chromatin Immunoprecipitation (ChIP), which are common applications for histone modification studies .

What are the optimal sample preparation protocols for using the 2-hydroxyisobutyryl-HIST1H2BC (K57) Antibody in immunofluorescence experiments?

For optimal immunofluorescence results with the 2-hydroxyisobutyryl-HIST1H2BC (K57) Antibody, the following protocol is recommended:

• Fixation: Fix cells in 4% formaldehyde for 10-15 minutes at room temperature.

• Permeabilization: Permeabilize using 0.2-0.5% Triton X-100 in PBS for 5-10 minutes.

• Blocking: Block non-specific binding with 1-5% normal serum (from the same species as the secondary antibody) for 30-60 minutes.

• Primary Antibody Incubation: Dilute the 2-hydroxyisobutyryl-HIST1H2BC (K57) Antibody at 1:10-1:100 and incubate overnight at 4°C .

• Secondary Antibody: Use fluorophore-conjugated anti-rabbit secondary antibodies at manufacturer-recommended dilutions.

This protocol is similar to that used for other histone modification antibodies such as Histone H2B (butyryl K5), which demonstrated successful staining in HeLa cells treated with sodium butyrate . For enhanced detection of 2-hydroxyisobutyryl modifications, pretreatment of cells with HDAC inhibitors or metabolic precursors may increase signal intensity.

How can researchers validate the specificity of 2-hydroxyisobutyryl-HIST1H2BC (K57) antibody signals in their experiments?

To validate the specificity of 2-hydroxyisobutyryl-HIST1H2BC (K57) antibody signals, researchers should implement multiple control experiments:

• Peptide Competition Assay: Pre-incubate the antibody with excess immunizing peptide before staining to block specific binding.

• Positive Control Treatment: Treat cells with sodium butyrate (30mM for 4 hours) or other compounds known to enhance histone acylation levels, similar to protocols used with other histone modification antibodies .

• Genetic Controls: Use CRISPR/Cas9 to generate K57R mutants (preventing modification at this site) and compare antibody signals.

• Cross-reactivity Testing: Test the antibody against other histone modifications at similar sites (K57 acetylation, methylation, or other acylations) to ensure specificity.

• Multiple Detection Methods: Confirm findings using at least two techniques (e.g., IF and Western blot).

• Mass Spectrometry Validation: For ultimate confirmation, immunoprecipitate the modified histone and analyze by mass spectrometry to confirm the presence of the 2-hydroxyisobutyryl modification at K57.

What are the recommended protocols for enhancing 2-hydroxyisobutyrylation levels in cultured cells for antibody validation and functional studies?

To enhance 2-hydroxyisobutyrylation levels in cultured cells, the following protocols are recommended:

• HDAC Inhibitor Treatment:

  • Sodium butyrate (NaBu): Treat cells with 5-30mM for 4-24 hours

  • Trichostatin A (TSA): 0.5-1μM for 12-24 hours

  • SAHA (Vorinostat): 1-5μM for 12-24 hours

• Metabolic Precursor Supplementation:

  • Add 2-hydroxyisobutyric acid (5-20mM) to culture media for 12-48 hours

  • Supplement with branched-chain amino acids (leucine, isoleucine) that serve as metabolic precursors

• Enzyme Modulation:

  • Overexpress putative 2-hydroxyisobutyryltransferases (similar to acetyltransferases)

  • Knockdown expression of known or predicted 2-hydroxyisobutyryl-lysine deacylases

• Metabolic Stress Conditions:

  • Glucose deprivation followed by refeeding (24h starvation, then 6h refeeding)

  • Hypoxic conditions (1-5% O₂) for 12-24 hours

The protocol should be optimized for each cell type, with validation of enhanced modification levels using techniques such as Western blotting, similar to methods used for other histone modifications .

How does the distribution of 2-hydroxyisobutyrylation at K57 of HIST1H2BC correlate with chromatin states and transcriptional activity?

The distribution of 2-hydroxyisobutyrylation at K57 of HIST1H2BC likely exhibits specific patterns related to chromatin states and transcriptional activity, though complete mapping studies are still emerging. Based on patterns observed with similar histone modifications:

• Genomic Localization: 2-hydroxyisobutyrylation at K57 may be enriched at:

  • Promoters of actively transcribed genes

  • Enhancer regions with high regulatory activity

  • Transcription start sites of developmentally regulated genes

• Correlation with Chromatin States:

  • Likely associated with euchromatic regions rather than heterochromatin

  • May co-occur with other active histone marks (H3K4me3, H3K27ac)

  • Potentially inversely correlated with repressive marks (H3K27me3, H3K9me3)

• Transcriptional Correlation:

  • The modification likely positively correlates with gene expression levels

  • May be dynamically regulated during transcriptional activation/repression

  • Could function in stabilizing transcription factor binding or recruitment of specific complexes

To definitively map these correlations, researchers should conduct ChIP-seq experiments with the 2-hydroxyisobutyryl-HIST1H2BC (K57) antibody and correlate findings with RNA-seq, ATAC-seq, and other histone modification ChIP-seq datasets, similar to approaches used for studying other histone marks .

What are the writer and eraser enzymes responsible for regulating 2-hydroxyisobutyrylation at K57 of HIST1H2BC, and how do they function?

The specific enzymatic machinery regulating 2-hydroxyisobutyrylation at K57 of HIST1H2BC is still being elucidated, but current research suggests:

Writer Enzymes (Potential 2-hydroxyisobutyryltransferases):

• P300/CBP: Known to catalyze multiple acylation reactions beyond acetylation

• PCAF/GCN5 family: May recognize and transfer 2-hydroxyisobutyryl groups

• MYST family enzymes: Potential catalytic activity for various acylations

Eraser Enzymes (2-hydroxyisobutyryl-deacylases):

• SIRT1-7: Particularly SIRT3, which has demonstrated deacylase activity beyond deacetylation

• HDAC1-11: Class I HDACs might have broader substrate specificity than previously recognized

Regulatory Mechanisms:

• Metabolic regulation: Cellular levels of 2-hydroxyisobutyryl-CoA likely influence modification rates

• Enzyme competition: Writer/eraser enzymes likely compete with machinery for other modifications

• Spatial regulation: Nuclear localization of metabolic intermediates may be controlled by dedicated transporters

To definitively identify these enzymes, researchers should conduct in vitro enzymatic assays with recombinant enzymes, followed by in vivo validation through genetic perturbation (CRISPR knockouts, catalytic mutants) combined with antibody detection of changes in 2-hydroxyisobutyrylation levels at K57 .

How does 2-hydroxyisobutyrylation at K57 interact with other histone modifications to create combinatorial regulatory effects?

2-hydroxyisobutyrylation at K57 likely participates in complex interplay with other histone modifications to create unique combinatorial effects:

• Cross-talk Mechanisms:

  • Competitive Inhibition: 2-hydroxyisobutyrylation may compete with other modifications (acetylation, methylation) at K57, creating mutually exclusive regulatory states

  • Sequential Modification: The presence of nearby modifications may enhance or inhibit 2-hydroxyisobutyrylation at K57

  • Allosteric Effects: Modifications at K57 might alter histone structure, affecting enzyme accessibility to other modification sites

• Combinatorial Reader Recognition:

  • Specific protein complexes may recognize patterns of modifications that include K57 2-hydroxyisobutyrylation

  • The combination of K57 2-hydroxyisobutyrylation with other modifications (e.g., H3K4me3) might create unique binding surfaces for specialized chromatin regulators

• Functional Outcomes:

  • Transcriptional regulation: Specific combinations may result in gene activation or repression

  • DNA repair pathway selection: Different modification combinations might recruit specific repair machinery

  • Replication timing: Combinations could influence origin firing and replication timing

To study these interactions, researchers should employ mass spectrometry-based approaches to identify co-occurring modifications, develop antibodies recognizing specific combinatorial patterns, and use genetic approaches to disrupt one modification while monitoring effects on others .

How should researchers interpret apparent discrepancies between 2-hydroxyisobutyryl-HIST1H2BC (K57) antibody signals in different experimental contexts?

When facing discrepancies in 2-hydroxyisobutyryl-HIST1H2BC (K57) antibody signals across different experimental contexts, researchers should consider:

• Technical Variables:

  • Antibody concentration effects: Titrate antibody across a broader range (1:1 to 1:1000)

  • Fixation method impacts: Compare paraformaldehyde, methanol, and acetone fixation

  • Antigen retrieval influence: Test with and without heat-induced epitope retrieval

  • Detection system sensitivity: Compare direct detection vs. amplification methods

• Biological Variables:

  • Cell cycle dependence: Synchronize cells and test at different cell cycle phases

  • Metabolic state influence: Compare nutrient-rich vs. nutrient-deprived conditions

  • Signal transduction effects: Test with and without specific pathway inhibitors

  • Cell density effects: Compare sparse vs. confluent cultures

• Integrated Analysis Approach:

  • Correlate antibody signals with orthogonal measurements of gene expression

  • Consider modification abundance in context of global histone modification levels

  • Examine temporal dynamics through time-course experiments

  • Validate key findings with genetic manipulation of suspected regulatory enzymes

When publishing, researchers should document all technical variables and present both representative and divergent results to advance understanding of context-dependent regulation of this modification .

What are the common technical challenges when using 2-hydroxyisobutyryl-HIST1H2BC (K57) antibody in ChIP experiments, and how can they be overcome?

When performing ChIP experiments with 2-hydroxyisobutyryl-HIST1H2BC (K57) antibody, researchers frequently encounter these challenges:

• Low Signal-to-Noise Ratio

  • Challenge: Background binding obscuring specific signals

  • Solution:

    • Increase washing stringency (higher salt concentration, 0.1-0.5% SDS)

    • Pre-clear chromatin with protein A/G beads

    • Use highly specific blocking agents (5% BSA, 5% milk)

    • Titrate antibody concentration (5-10 μg per ChIP reaction is typically optimal)

• Cross-Reactivity Issues

  • Challenge: Antibody binding to similar modifications

  • Solution:

    • Perform peptide competition controls

    • Include K57R mutant samples as negative controls

    • Compare ChIP-seq profiles with known distribution patterns of other marks

• Low Enrichment Efficiency

  • Challenge: Poor pulldown of modified histones

  • Solution:

    • Optimize crosslinking conditions (1% formaldehyde for 5-15 minutes)

    • Improve sonication for consistent chromatin fragments (200-500 bp)

    • Increase chromatin amount (20-40 μg per reaction)

    • Extend antibody incubation time (overnight at 4°C with rotation)

• Protocol Optimization Table:

Following these optimizations can significantly improve ChIP experiments targeting this specific modification, similar to approaches used for other histone modifications in ChIP protocols .

How can researchers integrate 2-hydroxyisobutyryl-HIST1H2BC (K57) antibody data with other omics datasets to develop comprehensive epigenetic models?

Integrating 2-hydroxyisobutyryl-HIST1H2BC (K57) antibody data with other omics datasets requires systematic analytical approaches:

• Multi-omics Data Integration Framework:

  • ChIP-seq Integration: Align 2-hydroxyisobutyryl-K57 peaks with:

    • Other histone modifications (H3K4me3, H3K27ac, H3K27me3)

    • Transcription factor binding sites

    • Chromatin accessibility (ATAC-seq)

    • DNA methylation patterns

  • Transcriptome Correlation:

    • Calculate enrichment scores of K57 2-hydroxyisobutyrylation at promoters

    • Correlate with RNA-seq expression values

    • Identify gene sets with coordinated regulation

    • Analyze temporal dynamics during cellular transitions

  • Proteome Connections:

    • Identify reader proteins that bind 2-hydroxyisobutyryl-K57 using pull-down MS

    • Map protein complexes associated with modified regions

    • Correlate with protein expression data

• Visualization and Modeling Approaches:

  • Generate multi-track genome browser views showing K57 2-hydroxyisobutyrylation in context

  • Develop machine learning models predicting gene expression from modification patterns

  • Create chromatin state segmentation including this modification

  • Build 3D chromatin interaction models incorporating modification data

This integrative approach will help researchers develop comprehensive models of how 2-hydroxyisobutyryl-K57 contributes to epigenetic regulation in different biological contexts .

What emerging technologies could enhance the study of 2-hydroxyisobutyryl-HIST1H2BC (K57) in single cells or at high spatial resolution?

Several cutting-edge technologies show promise for advancing 2-hydroxyisobutyryl-HIST1H2BC (K57) research:

• Single-Cell Epigenomics Approaches:

  • CUT&Tag in single cells: Adapting CUT&Tag for single-cell analysis with 2-hydroxyisobutyryl-HIST1H2BC (K57) antibody

  • Single-cell CUT&RUN: Providing higher resolution of modification distribution in heterogeneous populations

  • scChIC-seq: Chromatin immunocleavage sequencing adapted for single-cell resolution

  • Multimodal single-cell platforms: Simultaneous profiling of 2-hydroxyisobutyrylation and transcription

• Spatial Technologies:

  • Imaging mass cytometry: Using metal-conjugated antibodies for high-dimensional spatial mapping

  • Spatial transcriptomics with epigenetic markers: Correlating 2-hydroxyisobutyrylation with gene expression spatially

  • Super-resolution microscopy: Visualizing modification distributions at nanometer resolution

  • In situ sequencing of ChIP products: Mapping modifications directly in tissue contexts

• Real-Time Monitoring Systems:

  • Engineered reader domains with fluorescent reporters: Visualizing dynamics in living cells

  • FRET-based sensors: Detecting conformational changes associated with modification

  • Nanobody-based live imaging: Using small antibody fragments for real-time tracking

  • Optogenetic control of writer/eraser enzymes: Manipulating modification levels with spatiotemporal precision

These technologies will enable researchers to address questions about cell-type-specific regulation, spatial organization within nuclei, and dynamic changes in 2-hydroxyisobutyrylation during cellular processes that are currently inaccessible with bulk approaches .

How might 2-hydroxyisobutyryl-HIST1H2BC (K57) research contribute to understanding disease mechanisms and developing epigenetic therapies?

Research on 2-hydroxyisobutyryl-HIST1H2BC (K57) has significant potential for disease understanding and therapeutic development:

• Oncology Applications:

  • Biomarker Development: Changes in K57 2-hydroxyisobutyrylation patterns may serve as diagnostic or prognostic markers in specific cancer types

  • Therapeutic Targeting: Modulating enzymes controlling this modification could represent a novel intervention strategy

  • Resistance Mechanisms: Altered 2-hydroxyisobutyrylation may contribute to therapy resistance through chromatin reorganization

• Metabolic Disease Connections:

  • Metabolic-Epigenetic Interface: K57 2-hydroxyisobutyrylation likely responds to cellular metabolic state

  • Diabetes Research: Potential role in transcriptional dysregulation during insulin resistance

  • Obesity Models: May connect nutrient excess to altered gene expression programs

• Neurodegenerative Disorders:

  • Chromatin Stability: Potential role in maintaining neuronal genome integrity

  • Transcriptional Fidelity: May regulate expression of neuroprotective genes

  • Age-related Changes: Could be altered during brain aging and neurodegeneration

• Therapeutic Strategy Development:

  • Small Molecule Inhibitors: Design of specific inhibitors for enzymes regulating K57 2-hydroxyisobutyrylation

  • Metabolic Modulation: Dietary interventions affecting precursor availability

  • Combinatorial Approaches: Targeting multiple modifications simultaneously for synergistic effects

Research using the 2-hydroxyisobutyryl-HIST1H2BC (K57) antibody in disease models could reveal novel therapeutic targets and contribute to precision medicine approaches targeting the epigenome .