2-hydroxyisobutyryl-HIST1H2BC (K12) Antibody

What is the 2-hydroxyisobutyryl-HIST1H2BC (K12) Antibody and what biological process does it help investigate?

The 2-hydroxyisobutyryl-HIST1H2BC (K12) Antibody (PACO60519) is a polyclonal antibody generated in rabbits that specifically targets the 2-hydroxyisobutyryl post-translational modification on lysine 12 of histone variant HIST1H2BC. This antibody serves as a valuable tool for researchers studying histone modifications, which play critical roles in regulating gene expression, DNA replication, and repair processes .

Histone 2-hydroxyisobutyrylation is a relatively newly discovered modification that adds to the complexity of the histone code, contributing to chromatin structure regulation and transcriptional control. By recognizing this specific modification on K12 of HIST1H2BC, this antibody allows researchers to investigate its occurrence, distribution, and functional implications in various cellular contexts, particularly in epigenetics, cancer biology, and chromatin remodeling studies .

How does the HIST1H2BC protein function in chromatin organization?

HIST1H2BC (also known as Histone H2B type 1-C/E/F/G/I) serves as a core component of nucleosomes, the fundamental units of chromatin. Nucleosomes wrap and compact DNA into chromatin, thereby limiting DNA accessibility to cellular machineries that require DNA as a template . Each nucleosome consists of approximately 146 base pairs of DNA wrapped around an octamer of core histone proteins (two each of H2A, H2B, H3, and H4).

As a histone protein, HIST1H2BC plays a central role in transcription regulation, DNA repair, DNA replication, and chromosomal stability. The accessibility of DNA is regulated via a complex set of post-translational modifications of histones, collectively termed the "histone code," as well as through nucleosome remodeling . The 2-hydroxyisobutyryl modification at K12 of HIST1H2BC represents one such modification that contributes to this regulatory system.

What applications is the 2-hydroxyisobutyryl-HIST1H2BC (K12) Antibody validated for?

The 2-hydroxyisobutyryl-HIST1H2BC (K12) Antibody has been validated for multiple research applications, including:

This antibody demonstrates high specificity for human samples and can be used to detect the 2-hydroxyisobutyryl modification on HIST1H2BC K12 in various cell types and experimental contexts .

How should I design ChIP experiments using the 2-hydroxyisobutyryl-HIST1H2BC (K12) Antibody?

When designing chromatin immunoprecipitation (ChIP) experiments with the 2-hydroxyisobutyryl-HIST1H2BC (K12) Antibody, consider the following protocol:

• Cross-linking: Fix cells with 1% formaldehyde for 10 minutes at room temperature to preserve protein-DNA interactions.

• Chromatin preparation: Lyse cells and sonicate chromatin to fragments of 200-500 bp. Verify fragment size by agarose gel electrophoresis.

• Immunoprecipitation:

  • Pre-clear chromatin with protein A/G beads

  • Incubate cleared chromatin with 2-hydroxyisobutyryl-HIST1H2BC (K12) Antibody at 2-5 μg per reaction

  • Include appropriate controls: IgG negative control and a positive control antibody (e.g., anti-H3K4me3)

• Washing and elution: Perform stringent washes to remove non-specific binding, then elute protein-DNA complexes.

• Reverse cross-linking and DNA purification: Reverse formaldehyde cross-links and purify DNA for subsequent analysis.

• Analysis: Analyze precipitated DNA by qPCR, sequencing, or microarray to identify genomic regions associated with 2-hydroxyisobutyrylated HIST1H2BC K12.

For optimal results, validate antibody specificity using peptide competition assays or knockout/knockdown controls before proceeding with ChIP experiments . This method will allow you to map the genomic distribution of 2-hydroxyisobutyrylated HIST1H2BC K12 and correlate it with transcriptional states.

What cell treatment protocols enhance detection of 2-hydroxyisobutyryl modifications?

Treatment with sodium butyrate significantly enhances the detection of 2-hydroxyisobutyryl modifications in histone proteins. Based on experimental evidence:

• Sodium butyrate treatment: Incubate cells with 30 mM sodium butyrate for 4 hours prior to cell harvesting . This treatment inhibits histone deacetylases and promotes the accumulation of histone modifications, including 2-hydroxyisobutyrylation.

• Cell types validated: This enhancement effect has been demonstrated in several cell lines, including:

  • A549 (lung adenocarcinoma cells)

  • K562 (chronic myelogenous leukemia cells)

  • HepG2 (hepatocellular carcinoma cells)

  • 293 (human embryonic kidney cells)

• Detection method: Western blot analysis comparing untreated (-) versus treated (+) samples shows significantly enhanced signal intensity for 2-hydroxyisobutyryl-HIST1H2BC in treated samples .

• Alternative approaches: For studies focused on physiological regulation, consider using nutrient deprivation/refeeding protocols or metabolic inhibitors that alter cellular acyl-CoA pools, as these can naturally modulate 2-hydroxyisobutyrylation levels.

When implementing these treatments, include both treated and untreated controls to distinguish between basal and enhanced modification states, which will provide more comprehensive insights into the dynamics of this epigenetic mark.

How does 2-hydroxyisobutyrylation at K12 interact with other histone modifications on HIST1H2BC?

2-hydroxyisobutyrylation at K12 of HIST1H2BC exists within a complex network of histone modifications. Current research indicates several important interactions:

• Modification crosstalk: The presence of 2-hydroxyisobutyrylation at K12 may influence or be influenced by other nearby modifications, including:

  • Acetylation at neighboring lysine residues (K5, K15)

  • Methylation at various positions

  • Ubiquitination at K120

• Sequential modifications: Evidence suggests that certain modifications occur sequentially, with one modification serving as a prerequisite for another. For example:

  • Acetylation may precede 2-hydroxyisobutyrylation at specific sites

  • The presence of 2-hydroxyisobutyrylation may prevent other modifications at the same residue

• Combinatorial effects: The specific combination of modifications on HIST1H2BC creates a "modification signature" that can be recognized by reader proteins, leading to distinct functional outcomes .

• Reader protein specificity: Different effector proteins recognize specific modification patterns. The unique pattern including K12 2-hydroxyisobutyrylation may recruit distinct sets of chromatin regulators.

To investigate these interactions:

• Use sequential ChIP (re-ChIP) to identify genomic regions containing multiple specific modifications

• Employ mass spectrometry to identify co-occurring modifications on the same histone tail

• Perform in vitro binding assays to determine how different modification patterns affect reader protein recruitment

Understanding these interactions is crucial for deciphering the functional significance of 2-hydroxyisobutyrylation in chromatin regulation and gene expression.

What is the relationship between metabolic state and HIST1H2BC K12 2-hydroxyisobutyrylation?

Emerging research indicates a strong connection between cellular metabolism and histone 2-hydroxyisobutyrylation:

• Metabolic linkage: 2-hydroxyisobutyryl-CoA, the donor for this modification, is derived from branched-chain amino acid metabolism, particularly valine catabolism. Therefore, nutritional state and amino acid availability directly influence modification levels.

• Enzymatic regulation: While the specific enzymes catalyzing 2-hydroxyisobutyrylation at HIST1H2BC K12 are still being characterized, several classes of enzymes have been implicated:

  • Certain histone acetyltransferases (HATs) may possess 2-hydroxyisobutyryltransferase activity

  • Specific deacylases, including some sirtuins, may remove this modification

• Physiological conditions affecting modification:

  • Fasting/feeding cycles

  • Hypoxia

  • Cell proliferation state

  • Differentiation status

• Experimental approach: To investigate this relationship:

  • Culture cells in media with controlled carbon sources (glucose, galactose, glutamine)

  • Apply metabolic inhibitors targeting specific pathways

  • Perform metabolomic analysis in parallel with histone modification profiling

  • Use isotope tracing to track metabolic precursors to histone modifications

This metabolic connection suggests that 2-hydroxyisobutyrylation may serve as a mechanism linking cellular metabolism to epigenetic regulation, potentially explaining how environmental factors and nutrition can influence gene expression patterns through chromatin modifications.

What are common issues in Western blotting with 2-hydroxyisobutyryl-HIST1H2BC (K12) Antibody and how can they be resolved?

When performing Western blotting with the 2-hydroxyisobutyryl-HIST1H2BC (K12) Antibody, researchers may encounter several technical challenges. Here are common issues and their solutions:

Additionally, consider these optimization strategies:

• Extract histones using the acid extraction method to enrich for histones and their modified forms

• Include positive control samples (e.g., acid-extracted histones from sodium butyrate-treated cells)

• Use freshly prepared samples as histone modifications may be unstable during prolonged storage

• Incubate primary antibody overnight at 4°C for optimal binding

How can I validate the specificity of 2-hydroxyisobutyryl-HIST1H2BC (K12) Antibody for my experiments?

Validating antibody specificity is crucial for ensuring reliable experimental results. For the 2-hydroxyisobutyryl-HIST1H2BC (K12) Antibody, employ these validation strategies:

• Peptide competition assay:

  • Pre-incubate the antibody with increasing concentrations of:

    • The specific 2-hydroxyisobutyryl-K12 peptide (specific competitor)

    • Unmodified K12 peptide (negative control)

    • Peptides with other modifications at K12 (specificity control)

  • A reduction in signal only with the specific competitor confirms specificity

• Dot blot analysis:

  • Spot various modified and unmodified histone peptides on membrane

  • Probe with the antibody to assess cross-reactivity

  • Include peptides with similar modifications (acetylation, butyrylation) to test discrimination

• Immunoprecipitation-Mass Spectrometry:

  • Perform IP with the antibody

  • Analyze precipitated proteins by mass spectrometry

  • Confirm the presence of 2-hydroxyisobutyryl modification at K12

• Genetic approaches:

  • Use CRISPR/Cas9 to generate K12R mutants (prevents modification)

  • Compare antibody signal between wild-type and K12R mutants

  • Loss of signal in K12R mutants confirms specificity

• Treatment controls:

  • Compare samples from cells treated with/without sodium butyrate

  • Expected increase in signal after treatment supports functional specificity

• Cross-validation:

  • Compare results using alternative detection methods (e.g., mass spectrometry)

  • Use multiple antibodies targeting the same modification from different vendors if available

Thorough validation not only ensures experimental reliability but also advances the collective understanding of this modification's biological significance.

How should I integrate 2-hydroxyisobutyryl-HIST1H2BC K12 ChIP-seq data with other genomic datasets?

Integrating 2-hydroxyisobutyryl-HIST1H2BC K12 ChIP-seq data with other genomic datasets requires a systematic approach to derive meaningful biological insights:

• Data preparation and quality control:

  • Normalize ChIP-seq data to account for sequencing depth and input controls

  • Assess quality metrics: fragment size distribution, peak calling statistics, and replicate concordance

  • Generate browser-viewable files (bigWig) for visualization

• Integration with other histone modifications:

  • Compare with datasets for other HIST1H2BC modifications (K34, K108, K120)

  • Analyze co-occurrence or mutual exclusivity patterns

  • Identify unique chromatin states defined by modification combinations

• Correlation with transcriptional data:

  • Integrate with RNA-seq to correlate modification with gene expression

  • Analyze enrichment at promoters, gene bodies, and enhancers

  • Create heatmaps clustering genes by expression and modification levels

• Genomic feature analysis:

  • Compute enrichment at regulatory elements (promoters, enhancers, insulators)

  • Associate with chromatin accessibility data (ATAC-seq, DNase-seq)

  • Correlate with transcription factor binding sites (other ChIP-seq datasets)

• Analytical tools and approaches:

  • Use bedtools, deepTools, or HOMER for genomic overlaps and correlations

  • Apply machine learning algorithms to identify predictive patterns

  • Perform Gene Ontology or pathway analysis on genes associated with the modification

• Visualization strategies:

  • Generate genome browser tracks showing multiple datasets

  • Create metaplots centered on specific genomic features

  • Develop correlation matrices and heatmaps showing relationships between datasets

This integrated analysis will help position 2-hydroxyisobutyryl-HIST1H2BC K12 within the broader epigenomic landscape and provide insights into its functional role in chromatin organization and gene regulation.

What emerging technologies will advance our understanding of 2-hydroxyisobutyryl-HIST1H2BC modifications?

Several cutting-edge technologies are poised to transform our understanding of 2-hydroxyisobutyryl-HIST1H2BC modifications:

• Single-cell epigenomics:

  • Single-cell ChIP-seq to reveal cell-to-cell variability in modification patterns

  • Mass cytometry (CyTOF) with modification-specific antibodies to quantify modifications at single-cell resolution

  • Integration with single-cell transcriptomics to correlate modifications with gene expression heterogeneity

• Advanced imaging techniques:

  • Super-resolution microscopy to visualize the spatial distribution of modifications in the nucleus

  • Live-cell imaging using modification-specific nanobodies to track dynamics in real-time

  • Correlative light and electron microscopy to link modifications to chromatin ultrastructure

• Targeted protein engineering approaches:

  • CRISPR-based epigenome editing to install or remove specific modifications

  • Engineered reader domains to detect specific modification patterns

  • Synthetic histone proteins with defined modification patterns for functional studies

• Computational and systems biology methods:

  • Machine learning algorithms to predict modification sites and functional outcomes

  • Network analysis to identify regulatory hubs involving 2-hydroxyisobutyrylation

  • Integrative multi-omics approaches to place modifications in broader cellular context

• Structural biology advances:

  • Cryo-EM of nucleosomes with defined 2-hydroxyisobutyryl modifications

  • Structural studies of reader proteins bound to modified histone tails

  • Hydrogen/deuterium exchange mass spectrometry to analyze conformational changes induced by modifications

These technologies will enable researchers to move beyond correlative studies to establish causal relationships between specific modifications and biological outcomes, ultimately advancing our understanding of how the histone code regulates chromatin function.

How can computational approaches improve the analysis of 2-hydroxyisobutyryl-HIST1H2BC K12 data?

Computational approaches offer powerful tools for advancing research on 2-hydroxyisobutyryl-HIST1H2BC K12:

• Predictive modeling of modification sites:

  • Machine learning algorithms can predict potential 2-hydroxyisobutyrylation sites based on sequence context

  • These predictions can guide targeted experimental validation

  • Example approach: Train neural networks on known 2-hydroxyisobutyrylation sites to identify sequence and structural features associated with this modification

• Integrative multi-omics data analysis:

  • Develop computational frameworks to integrate ChIP-seq, RNA-seq, and proteomics data

  • Identify causal relationships between 2-hydroxyisobutyrylation and gene expression

  • Use Bayesian networks to model the complex relationships between multiple histone modifications

• Pattern recognition in genomic data:

  • Apply unsupervised learning to identify chromatin states defined by combinations of modifications

  • Develop pattern recognition algorithms to detect signature modification profiles

  • Example approach: Use self-organizing maps to cluster genomic regions by modification patterns, revealing functional domains

• Network analysis of modification-dependent interactions:

  • Construct protein-protein interaction networks centered on modified histones

  • Identify key nodes and regulatory hubs within these networks

  • Predict functional outcomes based on network perturbations

• Simulation of chromatin dynamics:

  • Develop molecular dynamics simulations incorporating 2-hydroxyisobutyryl modifications

  • Model how these modifications affect nucleosome stability and higher-order chromatin structure

  • Predict the impact of modifications on DNA accessibility

• Comparative genomics approaches:

  • Analyze conservation of modification sites across species

  • Identify evolutionarily conserved regulatory mechanisms

  • Leverage cross-species data to infer functional importance

These computational approaches not only enhance data analysis but also generate testable hypotheses that drive experimental design, creating a powerful cycle of computational prediction and experimental validation to advance our understanding of histone modifications.

What is a recommended workflow for studying 2-hydroxyisobutyryl-HIST1H2BC K12 in a new experimental system?

When investigating 2-hydroxyisobutyryl-HIST1H2BC K12 in a new experimental system, follow this comprehensive workflow:

• Preliminary Assessment and Validation:

  • Confirm expression of HIST1H2BC in your system using RT-qPCR or Western blotting

  • Validate the 2-hydroxyisobutyryl-HIST1H2BC K12 Antibody specificity in your cell type using methods described in FAQ 4.2

  • Establish baseline modification levels and optimize detection methods

• Modification Enhancement and Characterization:

  • Treat cells with sodium butyrate (30 mM for 4 hours) to enhance modification levels

  • Compare modification patterns in different cellular states (proliferation, differentiation, stress)

  • Perform time-course experiments to assess modification dynamics

• Genomic Distribution Analysis:

  • Conduct ChIP-seq to map genome-wide distribution of the modification

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

  • Correlate with transcriptional activity using RNA-seq

• Functional Investigation:

  • Disrupt the modification using site-specific mutagenesis (K12R substitution)

  • Manipulate enzymes responsible for adding/removing the modification

  • Assess phenotypic consequences of modification disruption

• Mechanistic Studies:

  • Identify proteins that recognize the modification using pull-down assays

  • Investigate downstream effects on chromatin structure and accessibility

  • Examine cross-talk with other histone modifications

• Integration and Contextual Analysis:

  • Place findings in the context of existing knowledge about histone modifications

  • Develop models explaining the functional role of this modification in your system

  • Identify system-specific characteristics or functions

This systematic approach provides a framework for comprehensive investigation of 2-hydroxyisobutyryl-HIST1H2BC K12 in any experimental system, from initial characterization through mechanistic understanding.

How can I effectively compare results across different studies using various 2-hydroxyisobutyryl-HIST1H2BC antibodies?

Comparing results across studies using different 2-hydroxyisobutyryl-HIST1H2BC antibodies requires careful consideration of several factors:

• Antibody characterization comparison:

  • Create a detailed catalog of antibodies used across studies, noting:

    • Epitope specificity (exact residue and surrounding sequence)

    • Host species and clonality (polyclonal vs. monoclonal)

    • Validation methods employed

    • Reported cross-reactivity with other modifications

  Antibody	Target Lysine	Validated Applications	Host/Clonality	Reference
  PACO60519	K12	ELISA, WB, ICC, IF	Rabbit/Polyclonal
  PACO60484	K34	ELISA, WB, ICC, IF	Rabbit/Polyclonal
  PACO59654	K108	ELISA, WB, IF	Rabbit/Polyclonal
  PACO60480	K120	ELISA, WB	Rabbit/Polyclonal

• Standardization approaches:

  • Perform direct antibody comparisons using identical samples

  • Include common positive controls across experiments

  • Normalize data to consistent reference points (e.g., total histone levels)

• Metadata collection and analysis:

  • Document experimental conditions in detail:

    • Cell types and culture conditions

    • Treatment protocols (concentration and duration)

    • Extraction and detection methods

  • Create a standardized format for reporting these variables

• Cross-validation strategies:

  • Confirm key findings using multiple antibodies

  • Supplement antibody-based detection with mass spectrometry

  • Validate with orthogonal approaches (mutagenesis, enzymatic modulation)

• Data integration techniques:

  • Use computational methods to normalize and integrate datasets

  • Identify consistent patterns across studies despite methodological differences

  • Meta-analysis approaches to quantitatively compare effect sizes

• Reporting standards:

  • Include detailed methods sections with antibody catalog numbers and concentrations

  • Provide representative images of full western blots

  • Share raw data in public repositories when possible