2-hydroxyisobutyryl-HIST1H2AG (K74) Antibody

What is 2-hydroxyisobutyryl-HIST1H2AG (K74) Antibody and what does it detect?

2-hydroxyisobutyryl-HIST1H2AG (K74) Antibody is a polyclonal antibody that specifically recognizes the 2-hydroxyisobutyrylation modification at lysine 74 (K74) of Histone H2A type 1 (HIST1H2AG). This antibody enables the detection and study of a specific post-translational modification involved in epigenetic regulation . The antibody binds to peptide sequences around the site of 2-hydroxyisobutyryl-Lys (74) derived from Human Histone H2A .

What is the difference between 2-hydroxyisobutyryl-HIST1H2AG antibodies targeting K74, K95, and K9?

These antibodies recognize the same histone protein (HIST1H2AG) but target different lysine residues that undergo 2-hydroxyisobutyrylation:

Different lysine residues may have distinct functional roles in gene regulation, making these antibodies complementary tools for comprehensive epigenetic research .

What applications is the 2-hydroxyisobutyryl-HIST1H2AG (K74) Antibody validated for?

The antibody has been validated for multiple experimental applications:

• Enzyme-Linked Immunosorbent Assay (ELISA) for quantitative detection

• Western Blotting (WB) at dilutions of 1:100-1:1000 for protein detection

• Immunocytochemistry (ICC) at dilutions of 1:500-1:1000 for cellular localization

These applications make it suitable for detection, quantification, and localization studies of 2-hydroxyisobutyrylated histone H2A in research settings.

What are the proper storage conditions for maintaining antibody activity?

For optimal antibody performance, follow these storage guidelines:

• Short-term storage (up to 2 weeks): Maintain refrigerated at 2-8°C

• Long-term storage: Store at -20°C or -80°C in small aliquots

• Avoid repeated freeze-thaw cycles as they can compromise antibody activity

• The antibody is supplied in a buffer containing 50% Glycerol and 0.03% Proclin 300 as preservatives

With proper storage, the antibody remains stable for approximately 12 months from the date of receipt .

How should I design my Western blot protocol using 2-hydroxyisobutyryl-HIST1H2AG (K74) Antibody?

For optimal Western blot results with this antibody:

• Sample preparation: Extract histones using acid extraction or commercial histone extraction kits

• Gel separation: Use 15-18% SDS-PAGE gels to properly resolve histone proteins

• Transfer: Employ PVDF membranes for optimal protein binding and signal

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

• Primary antibody incubation: Dilute 2-hydroxyisobutyryl-HIST1H2AG (K74) Antibody to 1:100-1:1000 in blocking buffer and incubate overnight at 4°C

• Washing: Wash membranes 3-5 times with TBST

• Secondary antibody: Use anti-rabbit IgG conjugated to HRP or fluorescent tags

• Detection: Develop using ECL or fluorescence-based detection systems

Recommended positive controls include human cell lines such as HeLa or A529, which express detectable levels of 2-hydroxyisobutyrylated histones .

What is the recommended protocol for immunocytochemistry using this antibody?

For immunocytochemistry applications:

• Cell preparation: Culture cells on coverslips and fix with 4% paraformaldehyde for 10 minutes

• Permeabilization: Treat with 0.2% Triton X-100 for 10 minutes

• Blocking: Block with 5% normal serum in PBS for 1 hour

• Primary antibody: Dilute 2-hydroxyisobutyryl-HIST1H2AG (K74) Antibody at 1:500-1:1000 and incubate overnight at 4°C

• Washing: Wash 3 times with PBS

• Secondary antibody: Apply fluorescently-labeled anti-rabbit secondary antibody for 1 hour at room temperature

• Nuclear counterstaining: Use DAPI for nuclear visualization

• Mounting and imaging: Mount with anti-fade medium and observe under fluorescence microscope

This protocol enables visualization of the nuclear localization pattern of 2-hydroxyisobutyrylated HIST1H2AG.

How can I validate the specificity of 2-hydroxyisobutyryl-HIST1H2AG (K74) Antibody in my experimental system?

To validate antibody specificity:

• Peptide competition assay: Pre-incubate the antibody with excess 2-hydroxyisobutyryl-K74 peptide before immunostaining; a specific antibody will show reduced or no signal

• Knockout/knockdown controls: Use CRISPR/Cas9 to delete HIST1H2AG or siRNA to knockdown its expression

• Mass spectrometry validation: Confirm the presence of 2-hydroxyisobutyrylation at K74 by immunoprecipitation followed by mass spectrometry

• Cross-reactivity testing: Test against similar histone modifications (e.g., acetylation, butyrylation) to ensure specificity

• Multiple antibody approach: Compare with other antibodies targeting the same modification at different lysine residues (K9, K95)

Validation approaches similar to those used for other histone modifications in the literature can help confirm the specificity of antibody binding to the 2-hydroxyisobutyrylated K74 residue.

How does 2-hydroxyisobutyrylation of HIST1H2AG compare with other histone post-translational modifications?

2-hydroxyisobutyrylation is a relatively newly discovered histone modification compared to acetylation, methylation, and phosphorylation. Key comparisons:

• Chemical structure: 2-hydroxyisobutyrylation involves addition of a bulkier group (2-hydroxyisobutyryl) compared to acetylation

• Genomic distribution: 2-hydroxyisobutyrylation can occur at multiple lysine residues (K9, K74, K95) on HIST1H2AG

• Functional roles: Emerging evidence suggests that 2-hydroxyisobutyrylation may have distinct roles in transcriptional regulation

• Regulatory enzymes: Writers and erasers of 2-hydroxyisobutyrylation are still being characterized, unlike the well-established HATs and HDACs for acetylation

• Cross-talk: 2-hydroxyisobutyrylation may interact with other histone modifications to form complex regulatory patterns

Understanding these differences is crucial for interpreting experimental results and designing studies to elucidate specific functions of HIST1H2AG 2-hydroxyisobutyrylation.

What are the recommended protocols for ChIP-seq using 2-hydroxyisobutyryl-HIST1H2AG (K74) Antibody?

For Chromatin Immunoprecipitation sequencing (ChIP-seq) applications:

• Crosslinking: Fix cells with 1% formaldehyde for 10 minutes at room temperature

• Chromatin preparation: Sonicate chromatin to fragments of 200-500 bp

• Immunoprecipitation:

  • Use 2-5 μg of 2-hydroxyisobutyryl-HIST1H2AG (K74) Antibody per IP reaction

  • Incubate with chromatin overnight at 4°C

  • Capture antibody-chromatin complexes with Protein A/G beads

• Washing: Perform stringent washes to remove non-specific binding

• Elution and reversal of crosslinks: Elute DNA and reverse crosslinks at 65°C

• Library preparation: Prepare sequencing libraries following standard protocols

• Validation: Validate enrichment by qPCR before sequencing

• Controls: Include input chromatin and IgG controls

This protocol allows genome-wide mapping of 2-hydroxyisobutyrylated HIST1H2AG (K74) binding sites to identify regulated genes and potential functional roles.

How can I integrate multiple histone modification analyses to understand the functional significance of HIST1H2AG K74 2-hydroxyisobutyrylation?

To comprehensively analyze the functional significance:

• Multi-omics approach:

  • Perform parallel ChIP-seq with antibodies targeting different modifications (K9, K74, K95 2-hydroxyisobutyrylation, acetylation, methylation)

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

  • Add ATAC-seq to assess chromatin accessibility

• Sequential ChIP (Re-ChIP):

  • Perform ChIP with one modification antibody followed by a second with another antibody

  • This determines co-occurrence of modifications on the same histone molecules

• Mass spectrometry-based quantification:

  • Enables detection of combinatorial modifications on the same histone tail

  • Provides stoichiometric information about modification abundance

• Machine learning integration:

  • Apply computational approaches to identify patterns and relationships between modifications

  • Predict functional outcomes based on modification combinations

This integrated approach provides deeper insights into how 2-hydroxyisobutyrylation at K74 contributes to the histone code and gene regulation.

What are common issues when using 2-hydroxyisobutyryl-HIST1H2AG (K74) Antibody and how can they be resolved?

Careful optimization of protocols for each experimental system is essential for obtaining reliable and reproducible results .

How do experimental conditions affect the detection of 2-hydroxyisobutyrylation in HIST1H2AG?

Several experimental factors can influence the detection of this modification:

• Cell culture conditions: Cell density, serum levels, and metabolic state can affect global 2-hydroxyisobutyrylation levels

• Extraction methods: Acid extraction is generally preferred for histone modifications to maintain their integrity

• Fixation protocols: Over-fixation may mask epitopes and reduce antibody binding

• Buffer composition: Presence of deacetylase inhibitors (e.g., sodium butyrate, TSA) may indirectly affect 2-hydroxyisobutyrylation levels

• Sample handling: Rapid processing minimizes enzymatic removal of modifications

• Detection systems: Enhanced chemiluminescence (ECL) vs. fluorescence-based detection may provide different sensitivity levels

Standardizing these conditions across experiments is crucial for reproducible detection and quantification of 2-hydroxyisobutyrylation patterns.

What are emerging research areas utilizing 2-hydroxyisobutyryl-HIST1H2AG (K74) Antibody?

The field is evolving rapidly, with several promising research directions:

• Metabolic regulation of 2-hydroxyisobutyrylation: Investigating how cellular metabolism influences this modification

• Dynamic regulation during development: Mapping changes in 2-hydroxyisobutyrylation patterns during cell differentiation and development

• Role in disease processes: Exploring alterations in 2-hydroxyisobutyrylation in cancer, neurodegenerative diseases, and other disorders

• Cross-talk with other epigenetic mechanisms: Understanding how 2-hydroxyisobutyrylation interacts with DNA methylation, chromatin remodeling, and other histone modifications

• Identification of reader proteins: Discovering proteins that specifically recognize and bind to 2-hydroxyisobutyrylated histones

• Development of selective inhibitors/modulators: Creating compounds that can specifically target the enzymes regulating this modification

These emerging areas represent opportunities for researchers to make significant contributions to epigenetic research using 2-hydroxyisobutyryl-HIST1H2AG (K74) Antibody.

How can 2-hydroxyisobutyryl-HIST1H2AG (K74) Antibody be used in single-cell epigenomic studies?

Integrating this antibody into single-cell techniques offers exciting possibilities:

• Single-cell CUT&Tag: Adapt CUT&Tag protocols using 2-hydroxyisobutyryl-HIST1H2AG (K74) Antibody to map this modification at single-cell resolution

• Mass cytometry (CyTOF): Develop metal-conjugated antibodies against 2-hydroxyisobutyrylated HIST1H2AG for high-dimensional single-cell analysis

• In situ hybridization combined with immunofluorescence: Simultaneously detect gene expression and 2-hydroxyisobutyrylation patterns in individual cells

• Live-cell imaging: Develop cell-permeable antibody fragments or nanobodies for tracking dynamics of 2-hydroxyisobutyrylation in living cells

• Spatial transcriptomics integration: Combine with spatial transcriptomics to correlate 2-hydroxyisobutyrylation patterns with gene expression in tissue contexts

These approaches would provide unprecedented insights into cell-to-cell variability in 2-hydroxyisobutyrylation patterns and their functional significance.