2-hydroxyisobutyryl-HIST1H2BC (K120) Antibody

What is 2-hydroxyisobutyryl-HIST1H2BC (K120) Antibody and what does it detect?

The 2-hydroxyisobutyryl-HIST1H2BC (K120) Antibody (PACO60480) is a polyclonal antibody produced in rabbits that specifically recognizes the 2-hydroxyisobutyryl modification at the lysine 120 residue of Histone H2B type 1-C/E/F/G/I (HIST1H2BC). This post-translational modification represents a relatively new addition to the known repertoire of histone marks that regulate chromatin structure and gene expression. The antibody enables researchers to study the presence, distribution, and functional significance of this specific histone modification in various biological contexts .

How does the 2-hydroxyisobutyryl modification differ from other histone modifications?

2-hydroxyisobutyrylation represents a distinct chemical modification compared to more well-characterized histone marks like acetylation, methylation, and phosphorylation. While acetylation neutralizes the positive charge of lysine residues, 2-hydroxyisobutyrylation adds a bulkier chemical group with specific structural properties that may create unique binding interfaces for chromatin-associated proteins. Unlike ubiquitylation, which plays established roles in transcription elongation and DNA replication , the complete functional repertoire of 2-hydroxyisobutyrylation is still being elucidated. Current research suggests it contributes to gene regulation through mechanisms that may partially overlap with but are distinct from other histone modifications .

What applications is this antibody validated for?

Based on available data, the 2-hydroxyisobutyryl-HIST1H2BC (K120) Antibody has been validated for the following applications:

*Dilutions extrapolated from similar 2-hydroxyisobutyryl antibodies . Researchers should perform optimization for their specific experimental systems.

The antibody has been shown to be effective in detecting 2-hydroxyisobutyrylation in human samples .

What is the difference between 2-hydroxyisobutyryl-HIST1H2BC (K108) and (K120) antibodies?

These antibodies recognize the same type of modification (2-hydroxyisobutyrylation) on the same histone protein (HIST1H2BC) but at different lysine residues. The K108 antibody (PACO59654) specifically detects modification at lysine 108, while the K120 antibody (PACO60480) recognizes modification at lysine 120 . These distinct modifications may have different biological functions, regulatory mechanisms, and genomic distributions. Using both antibodies in parallel experiments could provide insights into site-specific roles of 2-hydroxyisobutyrylation within the same histone protein.

What is the biological significance of studying histone 2-hydroxyisobutyrylation?

Histone 2-hydroxyisobutyrylation is emerging as an important epigenetic mark involved in gene regulation and chromatin structure . Similar to other histone modifications that create docking sites for regulatory proteins containing specific reader domains (such as bromodomains that recognize acetylated lysines) , 2-hydroxyisobutyrylation likely recruits specific effector proteins to facilitate downstream biological processes. Research into this modification is important for understanding epigenetic regulation and its role in various diseases, including cancer and neurological disorders . As part of the broader histone code, studying 2-hydroxyisobutyrylation contributes to our understanding of how cells regulate gene expression and maintain genomic integrity.

What are the optimal conditions for using this antibody in Western blotting experiments?

For optimal Western blotting results with the 2-hydroxyisobutyryl-HIST1H2BC (K120) Antibody:

• Sample preparation: Extract histones using specialized acid extraction protocols to ensure enrichment of histone proteins.

• Dilution: Start with a 1:500 dilution and adjust based on signal intensity .

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

• Primary antibody incubation: Incubate with diluted antibody overnight at 4°C.

• Secondary antibody: Use anti-rabbit IgG conjugated to HRP at 1:5000-1:10000 dilution.

• Positive controls: Include sodium butyrate-treated cell lysates, which can enhance histone modifications .

• Samples previously validated: Human cell lines including K562, HepG2, and 293 cell lysates have shown detectable signals .

When interpreting results, look for a band at approximately 14 kDa, corresponding to histone H2B, though the observed molecular weight may vary due to post-translational modifications .

How should this antibody be stored and handled to maintain its activity?

For optimal preservation of antibody activity:

When removing the antibody from storage, allow it to equilibrate at room temperature before opening to prevent condensation that could introduce contaminants.

What controls should be included when working with this antibody?

When designing experiments with the 2-hydroxyisobutyryl-HIST1H2BC (K120) Antibody, include the following controls:

• Positive control: Lysates from cell lines known to exhibit 2-hydroxyisobutyrylation at K120, such as human cell lines treated with sodium butyrate to enhance histone modifications .

• Negative controls:

  • Peptide competition assay using the immunizing peptide

  • Samples treated with deacetylase inhibitors but not 2-hydroxyisobutyrylation-promoting conditions

  • If available, samples from knockout models lacking the enzymes responsible for this modification

• Loading control: Use antibodies against total histone H2B or other core histones to normalize for histone content.

• Specificity control: Test cross-reactivity with other similar modifications by comparing with antibodies targeting other histone modifications like acetylation at the same or nearby residues.

Including these controls will help validate specificity and ensure the reliability of experimental results.

How can I optimize immunofluorescence experiments using this antibody?

For successful immunofluorescence studies:

• Fixation: Use 4% paraformaldehyde for 15 minutes at room temperature, followed by permeabilization with 0.1% Triton X-100.

• Antigen retrieval: Perform heat-mediated antigen retrieval using 10mM sodium citrate buffer (pH 6.0) to expose epitopes that may be masked during fixation.

• Blocking: Block with 5% normal serum from the species of the secondary antibody for 1 hour to reduce non-specific binding.

• Antibody dilution: Start with a 1:50 dilution and optimize as needed; the recommended range is 1:10-1:100 .

• Incubation conditions: Incubate primary antibody overnight at 4°C in a humidified chamber.

• Counterstaining: Use DAPI for nuclear staining, as 2-hydroxyisobutyrylation is a nuclear modification associated with chromatin .

• Mounting: Use anti-fade mounting medium to preserve fluorescence signal during imaging and storage.

Expected staining pattern should show nuclear localization with potential enrichment in specific nuclear regions depending on the biological context of the modification.

What methods can be used to validate the specificity of this antibody?

To ensure antibody specificity:

• Peptide competition assay: Pre-incubate the antibody with increasing concentrations of the immunizing peptide (containing 2-hydroxyisobutyrylated K120) before applying to samples. Signal reduction confirms specificity.

• Site-directed mutagenesis: Compare wildtype cells with those expressing a K120R mutant of HIST1H2BC that cannot be modified at this position.

• Mass spectrometry validation: Confirm the presence of 2-hydroxyisobutyrylation at K120 in immunoprecipitated samples using mass spectrometry.

• Cross-reactivity testing: Test against samples with other lysine modifications (acetylation, methylation) to ensure the antibody does not recognize these modifications.

• Dot blot analysis: Compare binding to modified versus unmodified peptides at various dilutions.

• Enzyme treatment: Treat samples with deacylases that remove 2-hydroxyisobutyryl groups and confirm signal reduction.

These validation steps are critical for confirming that experimental observations genuinely reflect the presence and dynamics of 2-hydroxyisobutyrylation at K120 of HIST1H2BC.

What is known about the relationship between 2-hydroxyisobutyrylation and other histone modifications?

Histone modifications often function as interconnected networks rather than isolated marks. While specific data on 2-hydroxyisobutyrylation at K120 is emerging, researchers should consider:

• Cross-talk with other modifications: Similar to how H2B ubiquitylation controls trans-methylation of histone H3 on lysines 4 and 79 , 2-hydroxyisobutyrylation may influence or be influenced by other histone marks.

• Competitive modifications: K120 can potentially undergo various modifications (acetylation, methylation, ubiquitylation), creating competition for the same residue.

• Sequential modifications: Some histone marks serve as prerequisites for others in sequential modification patterns.

• Genomic co-localization: ChIP-seq experiments can reveal whether 2-hydroxyisobutyrylation co-localizes with other histone marks associated with active transcription, repression, or DNA repair.

Researchers should design experiments that investigate potential synergistic or antagonistic relationships between 2-hydroxyisobutyrylation and well-characterized modifications like acetylation, which creates docking sites for bromodomain-containing proteins .

How can I design experiments to investigate the writers and erasers of 2-hydroxyisobutyrylation?

To identify and characterize the enzymes responsible for adding (writers) or removing (erasers) 2-hydroxyisobutyryl marks:

• Candidate approach: Test known histone acetyltransferases (HATs) and deacetylases (HDACs) for potential cross-activity with 2-hydroxyisobutyryl-CoA as a substrate using in vitro assays.

• Screening approach: Perform CRISPR/Cas9 or RNAi screens targeting chromatin-modifying enzymes and assess changes in global or specific 2-hydroxyisobutyrylation levels using the antibody.

• Biochemical purification: Use immobilized 2-hydroxyisobutyrylated peptides as bait to capture interacting proteins, followed by mass spectrometry identification.

• Metabolic regulation: Investigate connections between cellular metabolism and 2-hydroxyisobutyrylation, as the modification likely requires 2-hydroxyisobutyryl-CoA derived from cellular metabolic pathways.

• In vitro reconstitution: Test candidate enzymes using recombinant histones or nucleosomes and 2-hydroxyisobutyryl-CoA as substrate.

Understanding the enzymatic regulation of this modification will provide insights into its biological functions and potential as a therapeutic target.

What genomic regions are enriched for 2-hydroxyisobutyryl-HIST1H2BC (K120)?

While specific genomic distribution data for 2-hydroxyisobutyryl-HIST1H2BC (K120) is still emerging, researchers can use ChIP-seq approaches to map this modification genome-wide:

• Design experiments to compare its distribution with:

  • Actively transcribed genes (like H2B ubiquitylation, which is enriched in coding regions )

  • Replication origins (where H2B ubiquitylation has been detected )

  • Enhancers and promoters

  • Specific chromatin states (heterochromatin vs. euchromatin)

• Analyze co-occurrence with transcription factors or chromatin remodelers.

• Compare its distribution during different cell cycle phases or differentiation stages.

• Consider analyzing its presence at origin recognition complex (ORC) binding sites, as H2B modifications have been found at replication origins even in ORF-free regions .

These approaches will help establish the functional genomic context of this modification and inform hypotheses about its biological roles.

How can I integrate 2-hydroxyisobutyrylation data with other epigenomic datasets?

For comprehensive epigenomic analysis:

• Multi-omics integration: Combine ChIP-seq data for 2-hydroxyisobutyryl-HIST1H2BC (K120) with:

  • RNA-seq to correlate with gene expression

  • ATAC-seq or DNase-seq for chromatin accessibility

  • ChIP-seq for other histone modifications and transcription factors

  • DNA methylation data

• Computational approaches:

  • Use correlation analysis to identify relationships with other epigenetic marks

  • Apply machine learning algorithms to identify predictive patterns

  • Employ network analysis to understand regulatory interactions

• Visualization tools:

  • Genome browsers for region-specific visualization

  • Heatmaps for global pattern recognition

  • Principal component analysis for dimension reduction and pattern detection

• Functional genomics validation:

  • Target specific regions with CRISPR-based epigenome editing

  • Validate computational predictions with directed experiments

This integrated approach will provide a more complete understanding of how 2-hydroxyisobutyrylation fits within the broader epigenetic landscape and contributes to genomic regulation.

Why might I see unexpected bands or patterns in Western blots using this antibody?

Multiple bands or unexpected patterns when using 2-hydroxyisobutyryl-HIST1H2BC (K120) Antibody could result from several factors:

• Multiple histone variants: The H2B family includes multiple variants with similar sequences that might be recognized if the epitope is conserved.

• Cross-reactivity: The antibody may recognize similar modifications or sequences in other proteins. Always validate specificity using controls.

• Degradation products: Histone proteins may degrade during sample preparation, leading to lower molecular weight bands.

• Mobility shifts due to modifications: As noted for HIST1H2BA, "The actual band is not consistent with the expectation... The mobility is affected by many factors... different modified forms at the same time [may cause] multiple bands" .

• Incomplete denaturation: Ensure complete denaturation of protein complexes by adequate heating in sample buffer.

• Post-translational modifications: Additional modifications can alter protein migration. The calculated MW for H2B is approximately 14 kDa, but observed MW may vary .

• Non-specific binding: Optimize blocking conditions and antibody dilutions to reduce background.

What factors might affect the detection of 2-hydroxyisobutyrylation in biological samples?

Several factors can influence detection sensitivity and specificity:

• Modification abundance: 2-hydroxyisobutyrylation may be present at low levels in certain cell types or conditions, requiring enrichment strategies.

• Cell cycle stage: Histone modifications often exhibit cell cycle-dependent patterns, as observed with H2B ubiquitylation during S phase .

• Fixation artifacts: Overfixation can mask epitopes, while underfixation may result in signal loss. Optimize fixation protocols for each application.

• Enzyme activity in lysates: Deacylases in lysates may remove modifications during sample preparation. Include deacylase inhibitors in lysis buffers.

• Treatment conditions: Sodium butyrate treatment (30mM for 4 hours) has been shown to enhance detection of some histone modifications .

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

• Technical variation: Standardize sample collection, processing times, and storage conditions to reduce technical variability.

Understanding these factors will help researchers optimize experimental conditions and correctly interpret results.

How can I quantify changes in 2-hydroxyisobutyrylation levels?

For accurate quantification of 2-hydroxyisobutyrylation levels:

• Western blot quantification:

  • Use digital imaging systems rather than film for linear dynamic range

  • Include a standard curve of recombinant or synthetic modified peptides

  • Normalize to total H2B levels using a modification-insensitive H2B antibody

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

• Mass spectrometry approaches:

  • Employ stable isotope labeling with amino acids in cell culture (SILAC)

  • Use selected reaction monitoring (SRM) or parallel reaction monitoring (PRM)

  • Add synthetic peptide standards for absolute quantification

• ChIP-seq quantification:

  • Use spike-in controls for normalization between samples

  • Apply appropriate statistical methods for differential binding analysis

  • Validate changes at selected loci using ChIP-qPCR

• Immunofluorescence quantification:

  • Use automated image analysis with consistent thresholding

  • Measure nuclear intensity relative to DAPI or total H2B staining

  • Analyze sufficient cell numbers for statistical significance

Whichever method is chosen, include appropriate controls and statistical analysis to ensure reliable quantification of biological changes.

How do I determine if changes in 2-hydroxyisobutyrylation are cause or consequence of observed phenotypes?

Establishing causality requires experimental approaches that go beyond correlation:

• Genetic manipulation:

  • Generate K120R mutants that cannot be modified at this position

  • Create fusion proteins that mimic constitutive modification

  • Use CRISPR/Cas9 to edit the endogenous locus

• Temporal analysis:

  • Perform time-course experiments to determine if modification changes precede phenotypic changes

  • Use rapid induction or inhibition systems to analyze immediate effects

• Targeted modification:

  • Apply CRISPR-based epigenome editing to selectively add or remove modifications at specific loci

  • Use domain-focused approaches that target specific genomic regions

• Rescue experiments:

  • Test if restoring the modification reverses phenotypes in deficient models

  • Use complementation with wildtype or mutant histones in knockout backgrounds

• Mechanism dissection:

  • Identify and manipulate downstream effectors that recognize the modification

  • Create separation-of-function mutations that affect specific downstream pathways

These approaches will help distinguish whether 2-hydroxyisobutyrylation is a driver or consequence of the biological processes being studied.

What considerations are important when comparing results across different studies of histone modifications?

When comparing studies or designing new experiments:

• Antibody considerations:

  • Different antibodies may have varying specificities and sensitivities

  • Some studies may use different clones or lots with different properties

  • Validation methods may vary between studies

• Experimental conditions:

  • Cell types, culture conditions, and treatments affect modification levels

  • Fixation methods and durations influence epitope accessibility

  • Sample preparation protocols may preserve modifications differently

• Technical approaches:

  • ChIP-seq protocols vary in crosslinking, sonication, and library preparation

  • Mass spectrometry approaches differ in sensitivity and coverage

  • Western blot quantification methods may use different normalization strategies

• Data analysis:

  • Bioinformatic pipelines employ different algorithms and parameters

  • Statistical thresholds for significance vary between studies

  • Definition of enriched regions or peaks may differ

• Biological context:

  • Cell cycle stage impacts histone modification patterns

  • Cellular differentiation state affects the epigenetic landscape

  • Stress conditions can dramatically alter modification profiles

Researchers should explicitly state these variables when publishing and consider them when interpreting results across studies.

What are emerging applications for studying 2-hydroxyisobutyrylation in disease models?

As with other histone modifications that contribute to epigenetic dysregulation in disease, 2-hydroxyisobutyrylation represents a promising area for investigating:

• Cancer biology: Examine how altered 2-hydroxyisobutyrylation patterns contribute to oncogenic gene expression programs and chromosomal instability.

• Neurodegenerative disorders: Investigate potential roles in transcriptional dysregulation associated with conditions like Alzheimer's and Parkinson's diseases.

• Metabolic disorders: Explore connections between metabolic state, 2-hydroxyisobutyryl-CoA availability, and chromatin regulation.

• Development and differentiation: Study how this modification changes during cellular differentiation and development.

• Aging research: Analyze age-associated changes in 2-hydroxyisobutyrylation patterns across tissues.

• Inflammatory conditions: Examine potential roles in regulating inflammatory gene expression programs.

Researchers can leverage the 2-hydroxyisobutyryl-HIST1H2BC (K120) Antibody to investigate these disease contexts, potentially identifying novel biomarkers or therapeutic targets.

How can single-cell approaches advance our understanding of 2-hydroxyisobutyrylation?

Single-cell technologies offer exciting opportunities for studying epigenetic heterogeneity:

• Single-cell CUT&Tag or CUT&RUN: Adapt these techniques to map 2-hydroxyisobutyrylation across individual cells.

• scATAC-seq integration: Correlate chromatin accessibility with 2-hydroxyisobutyrylation patterns.

• Multimodal single-cell analysis: Simultaneously profile gene expression and histone modifications.

• Live-cell imaging: Develop fluorescent sensors for real-time tracking of 2-hydroxyisobutyrylation dynamics.

• Single-cell proteomics: Emerging mass cytometry approaches might enable quantification of histone modifications at single-cell resolution.